Method for altering the nutritional content of plant seed

ABSTRACT

The invention provides preselected DNA sequences and methods of using them to alter the nutritional content of plant seed. Methods of the invention are directed to increasing the weight percent of an amino acid essential to the diet of animals, or increasing the starch content, of a plant. One method involves stably transforming a cell of a plant with a preselected DNA sequence encoding an RNA molecule substantially identical or complementary to a messenger RNA encoding a plant seed storage protein. An alternative method employs stably transforming cells with at least two preselected DNA sequences, one of which encodes an RNA molecule substantially identical or complementary to a messenger RNA (mRNA) encoding a plant seed storage protein, and the other preselected DNA molecule which encodes a preselected polypeptide. The transformed cells are used to generate fertile transgenic plants and seeds, which are characterized by expression of the preselected DNA sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 08/763,704filed Dec. 9, 1996, now U.S. Pat. No. 6,326,527 which is acontinuation-in-part of U.S. application Ser. No. 08/112,245 filed Aug.25, 1993 now abandoned, the disclosures of which are incorporated byreference.

FIELD OF THE INVENTION

The invention relates generally to modification of the nutritionalcontent of maize seed utilizing preselected DNA constructs. Morespecifically, the invention relates to the use of preselected DNAconstructs to transform maize plants so as to alter the levels ofproteins, such as seed storage proteins, e.g., the prolamines (zeins),in the seeds of transgenic maize plants. Thus, the invention provides amechanism to replace nutritionally deficient proteins with nutritionallyenhanced proteins, and/or to increase the levels of starch, in the seedof transgenic maize plants.

BACKGROUND OF THE INVENTION

In agriculturally important seed crops, the expression of storageprotein genes directly affects the nutritional quality of the seedprotein. In maize, the prolamine (zein) fraction of storage proteinscomprises over 50% of the total protein in the mature seed. The zeinsdesignated α-zein are especially abundant. The α-zein polypeptidescontain extremely low levels of the essential amino acids lysine andtryptophan. Thus, maize seed protein is deficient in these amino acidsbecause such a large proportion of the total seed storage protein iscontributed by the α-zeins (Mertz et al., 1964).

The development of breeding steps to improve maize based on themanipulation of zein profile is hampered by the complexity of the zeinproteins. The term “zein” encompasses a family of some 100 relatedproteins. Zeins can be divided into four structurally distinct types:α-zeins include proteins with molecular weights of 19,000 and 22,000daltons; β-zeins include proteins with a molecular weight of 14,000daltons; γ-zeins include proteins with molecular weights of 27,000 and26,000 daltons; and δ-zeins include proteins having a molecular weightof 10,000 daltons. The α-zeins are the major zein proteins found in theendosperm of maize kernels. However, the complexity of zein proteinsgoes beyond these size classes. Protein sequence analyses indicates thatthere is microheterogenicity in zein amino acid sequences. This is inaccord with isoelectric focusing analyses which show charge differencesin zein proteins. Over 70 genes encoding the zein proteins have beenidentified (Rubenstein, 1982), and the zein genes appear to be locatedon at least three chromosomes. Thus, the zein proteins are encoded by amultigene family.

Based on sequence and hybridization data, the zein multigene family isdivided into several subfamilies. Each subfamily is defined by sequencehomology to a cDNA clone: A20, A30, B49, B59, or B36. Hybrid-selecttranslation studies which employ B49 and B36 select mRNAs that code forpredominantly heavy class (23 kD) α-zein proteins, while A20, A30, andB59 select for predominantly the light class (19 kD) α-zein proteins(Heidecker and Messing, 1986). A comparison of zein sequences in each ofthe subfamilies A20, A30 and B49 have identified four distinctfunctional domains (Messing et al., 1983). Region I corresponds to thesignal peptide present in most, if not all, zeins. Regions II and IVcorrespond to the amino and carboxyl termini, respectively, of themature zein protein. Region III corresponds to the coding region betweenRegions II and IV, including a region which has tandem repeats of a 20amino acid sequence.

There are several mutations known to cause reductions in zein synthesisthat lead to alterations in the amino acid content of the seed. Forexample, in the seeds of plants homozygous for the recessive mutationopaque-2, the zein content is reduced by approximately 50% (Tsai et al.,1978). The opaque-2 mutation primarily affects synthesis of the 19 and22 kD α-zein proteins, causing a significant decrease in the level ofthe 19 kD zein fraction and reducing the accumulation of the 22 kD zeinfraction to barely detectable levels (Jones et al., 1977). In thismutant, there is a concomitant increase in the proportion of morenutritionally balanced proteins, e.g., albumins, globulins andglutelins, deposited in the seed. The net result of the altered storageprotein patterns is an increase in the essential amino acids lysine andtryptophan in the mutant seed (Misra et al., 1972).

Two other recessive mutations, floury-2 and sugary-1, result inincreased levels of methionine in the seed. The increased methioninecontent in the seeds of floury-2 mutants is the result of a decrease inthe zein/glutelin ratio, due to reductions in the levels of both the 19and 22 kD α-zein fractions, and an apparent increase in the methioninecontent of the glutelin fraction (Hansel et al., 1973; Jones, 1978). Insugary-1 mutants, there is a decrease in zein synthesis coupled with anincrease in the methionine content of the zein and glutelin fractions(Paulis et al., 1978).

As demonstrated by the opaque-2, floury-2, and sugary-1 mutations,reductions in zein synthesis and/or changes in the relative proportionsof the storage protein fractions can affect the overall amino acidcomposition of the seed. Unfortunately, poor agronomic characteristics(kernel softness, reduced yield, lowered resistance to disease) areassociated with the opaque and floury mutations, preventing their readyapplication in commercial breeding.

Another way that genes can be down regulated in animals and plantsinvolves the expression of antisense genes. A review of the use ofantisense genes in manipulating gene expression in plants can be foundin van der Krol et al. (1988a;1988b). The inhibition ot expression ofseveral endogenous plant genes has been reported. For example, U.S. Pat.No. 5,107,065 discloses down regulation of polygalacturonase activity byexpression of an antisense gene. Other plant genes down regulated usingantisense genes include the genes encoding chalcone synthase and thesmall subunit of ribulose-1,5-biphosphate carboxylase (van der Krol etal., 1988c; Rodermel et al., 1988). However, to date there has been nodescription of attempts to use antisense technology to alter thenutritional content of seeds.

Down regulation of gene expression in a plant may also occur throughexpression of a particular transgene. This type of down regulation isreferred to as co-suppression and involves coordinate silencing of atransgene and a second transgene or a homologous endogenous gene (Matzkeand Matzke, 1995). For example, cosuppression of a herbicide resistancegene in tobacco (Brandle et al., 1995), polygalacturonidase in tomato(Flavell, 1994) and chalcone synthase in petunia (U.S. Pat. No.5,034,323) have been demonstrated. Flavell (1994) suggested thatmulticopy genes, or gene families, must have evolved to avoidcosuppression in order for multiple copies of related genes to beexpressed in a plant.

Thus, there is a need for a method to alter the nutritional content ofseeds and produce kernels with good agronomic characteristics, includingmaintaining kernel hardness, yield, and disease resistance of the parentgenotype. Furthermore, there is a need for a method to decreaseexpression of seed storage proteins of poor nutritional quality whileincreasing proteins with higher contents of nutritionally advantageousamino acids, such as methionine and lysine, and/or while increasing thestarch content of seeds.

SUMMARY OF THE INVENTION

The invention provides methods which employ a genetically engineered,preselected DNA sequences or segments to alter the nutritional contentof plant seeds. The expression of said preselected DNA sequence resultsin an altered protein and/or amino acid composition in the transgenicplant, plant tissue, plant part, or plant cell relative to thecorresponding nontransformed, i.e., nontransgenic, plant, plant tissue,plant part, or plant cell. Preferably, the seeds of said transgenicplant have an increased amount, e.g., weight percent, of at least oneamino acid essential to the diet of animals, relative to nontransformed,i.e., nontransgenic, seeds. An increase in the weight percent of atleast one amino acid essential to the diet of animals, e.g., lysine,methionine, isoleucine, tryptophan, or threonine, in seeds increases thenutritional value of those seeds for animal, e.g., feeds for poultry andswine, or human consumption.

Thus, the invention provides a method which comprises stablytransforming cells of a plant with an expression cassette. Theexpression cassette comprises a preselected DNA sequence which codes foran RNA molecule which is substantially identical (sense), orcomplementary (antisense), to all or a portion of a messenger RNA(“target” mRNA), i.e., an endogenous or “native” mRNA, which is presentin an nontransformed plant cell. The target mRNA encodes a plant seedstorage protein, preferably a protein which is deficient in at least oneamino acid, and more preferably deficient in an amino acid which isessential to the diet of animals.

The resultant transformed cells are used to regenerate fertiletransgenic plants which in turn yield transgenic seeds, wherein thepreselected DNA sequence is expressed in the transgenic seeds in anamount effective to substantially reduce or decrease the amount, weightpercent or level of a seed storage protein relative to the amount,weight percent or level of said seed storage protein present in thecorresponding nontransgenic seeds, e.g., seeds of a nontransformed ROcontrol plant or corresponding nontransformed seeds isolated from thetransgenic plant. The seed storage protein is one which is deficient inat least one amino acid essential to the diet of an animal. Preferably,the decrease in the amount of the seed storage protein results in anincrease in the weight percent of seed storage proteins comprisinghigher percentages of nutritionally advantageous amino acids. Thepreselected DNA sequence preferably codes for an RNA moleculesubstantially complementary to all or a portion of a mRNA coding for a19 kD or 22 kD α-zein protein. A reduction in seed storage proteins,e.g., the α-zeins, may be accompanied by a decrease in the degree ofkernel hardness. Hardness of the kernel may be enhanced in these casesby modification of the kernel phenotype as described for the opaque-2mutation (Lopes and Larkins, 1991) or by genetically modifying plants toincrease the levels of certain endosperm proteins such as the 27 kDγ-zein.

The genetically engineered DNA sequences of the invention are“preselected” in that the coding regions contained therein have beenisolated in vitro, and identified at least functionally. Thus, a“preselected” DNA is a DNA sequence or segment that has been isolatedfrom a cell, purified, and amplified. The choice of the preselected DNAsequence will be based on the amino acid composition of the polypeptideencoded by the sense strand of a preselected DNA sequence, andpreferably, the ability of the polypeptide to accumulate in seeds.Preferably, the number of said coding regions has also been ascertained.Also preferably, the isolated DNA molecule is “recombinant” in that itcontains preselected DNA sequences from different sources which,preferably, have been linked to yield chimeric expression cassettes. Thepreselected DNA sequences are preferably about 2-3 kb.

The invention further provides a method to increase the starch contentof a plant, plant part, plant tissue or plant cell. The method comprisesstably transforming cells of a plant with an expression cassette. Theexpression cassette comprises a preselected DNA sequence coding for anRNA molecule substantially identical, or complementary, to all or aportion of at least one mRNA coding for a plant seed storage protein.Preferably, the preselected DNA sequence is operably linked to apromoter functional in a plant and/or seed. Transformed cells are usedto regenerate fertile transgenic plants and seeds. The preselected DNAsequence is preferably expressed in the transgenic seeds in an amounteffective to decrease the weight percent of seed storage protein in thetransgenic seed over the weight percent of seed storage protein presentin the corresponding nontransgenic seed. The preselected DNA sequence isalso preferably expressed in the transgenic seeds in an amount effectiveto increase the weight percent of starch in the transgenic seed over theweight percent of starch present in the corresponding nontransgenicseed. An increase in the weight percent of the starch of seeds improvesthe food value of the seed, or its value as a source of starch for usein processed food products or in various industrial applications.Moreover, an increase in starch content in transgenic seeds can resultin an increase in the starch recovered from those seeds.

Also provided is a method to inhibit a family or subfamily of seedstorage proteins. Seed storage proteins such as the zein proteins ofmaize are encoded in a multigene family. Portions of the amino acidsequence of, and DNA sequences encoding, seed storage proteins in agiven family share amino acid, and DNA, sequence homology, respectively(termed “family”-specific sequences). Other portions of the amino acidsequence of, and DNA sequences encoding, a zein seed storage protein ina subfamily share amino acid, and DNA, sequence homology, respectively,with one another (termed “subfamily”-specific sequences). A preselectedDNA sequence corresponding to family-, or subfamily-, specific sequencescan be employed to inhibit the production of a family or subfamily ofzein proteins. An expression cassette is provided which comprises apreselected DNA sequence encoding an RNA molecule which is substantiallyidentical, or complementary, to all or a portion of a mRNA that issubstantially homologous in sequence among members of a family orsubfamily of zein proteins. The expression cassette which comprises thepreselected DNA sequence is then introduced into plant cells, which areregenerated to yield transgenic plants and seeds. The transgenic seedsare characterized by substantial inhibition of a preselected family orsubfamily of seed storage protein. In a preferred embodiment, thepreselected DNA sequence encodes an RNA molecule which is substantiallycomplementary to all or a portion of a mRNA coding for a 20 amino acidsequence which is present in multiple, tandem copies in the A20subfamily of the α-zein proteins.

Another embodiment of the invention comprises plant cells, plant tissue,plant parts or plants stably transformed with at least two preselectedDNA sequences. The first preselected DNA sequence encodes an RNAmolecule substantially identical, or complementary, to all or a portionof a mRNA encoding a seed storage protein, e.g., an endogenous seedstorage protein, preferably one which is relatively deficient in atleast one amino acid essential to the diet of animals compared to otherseed storage proteins. The second preselected DNA sequence encodes apolypeptide of desired amino acid composition, i.e., a polypeptidecomprising at least one amino acid essential to the diet of animals. Thepolypeptide, preferably, has physical properties which minimizedisruption of seed cellular structure and therefore grain quality. It ispreferred that each preselected DNA sequence is operably linked to apromoter functional in a plant and/or seed.

Following transformation, transformed plant cells having the first andsecond preselected DNA sequences stably, i.e., chromosomally, integratedinto their genome are selected and used to regenerate fertile transgenicplants and seeds. The transgenic seeds are characterized by theexpression of the first DNA sequence in an amount effective tosubstantially reduce or decrease the amount, weight percent, or level,of the undesirable seed storage protein, or an amino acid present insaid protein, over the amount, weight percent, or level, of that seedstorage protein, or the amino acid present in that protein, which ispresent in nontransgenic seeds. The transgenic seeds are also preferablycharacterized by the expression of the second DNA sequence as a plantprotein in an amount effective to yield an increase in the amount,weight percent or level of at least one amino acid essential to the dietof animals over the amount, weight percent or level of that amino acidpresent in nontransgenic seeds.

In a preferred embodiment, the expression of the first preselected DNAsequence in transgenic maize seed inhibits the weight percent of 19 kDor 22 kD α-zein. In another preferred embodiment, the expression of thesecond preselected DNA sequence in transgenic seed results in anincrease in the weight percent of a 10 kD δ-zein protein. In yet anotherpreferred embodiment, the expression of the second preselected DNAsequence in transgenic seed results in an increase in the weight percentof a 27 kD zein protein. In yet another preferred embodiment, the secondpreselected DNA encodes a synthetic polypeptide, such as MB1 (Beauregardet al., 1995). MB1 is a stable synthetic polypeptide highly enriched inamino acids essential for animal nutrition (e.g., methionine, threonine,lysine, and leucine) which also adopts an α-helical conformation. Thesynthetic polypeptide MB1 shares some properties of maize zein proteins,e.g., MB1 is alcohol soluble and contains multiple α-helical domains.However, other polypeptides, synthetic and naturally occurring, withpreselected desired amino acid compositions, and genes coding therefor,could be employed in the practice of the invention. As used herein, theterm “polypeptide” includes protein.

The invention also provides a method to increase the amount, weightpercent or level of a polypeptide in a plant. The method comprisesstably transforming plants, plant cells, plant tissue or plant partswith a first preselected DNA sequence which encodes a seed storageprotein and a second preselected DNA sequence which encodes at least aportion of a preselected, desired polypeptide. The polypeptide may beencoded by the genome of the nontransformed plant or plant cell(“endogenous” or “native”), or, alternatively may not be native to,i.e., present in, the genome of the nontransformed “wild type” plant orplant cell (termed “heterologous,” “non-native” or “foreign”).Preferably, the second preselected DNA sequence encodes a bacterialenzyme, e.g., AK, DHDPS, EPSPS, a bacterial toxin, e.g., the crystaltoxin from Bt, a seed storage protein, e.g., Z27, or a non-maize seedstorage protein, such as nut and legume seed storage proteins. See, forexample, U.S. Pat. No. 4,769,061; U.S. Pat. No. 4,971,908;PCT/US90/04462; PCT/WO89/11789; and Altenbach et al. (1989).

Transformed plant cells having the first and second preselected DNAsequences stably, i.e., chromosomally, integrated therein are selectedand used to regenerate fertile transgenic plants and seeds. Transgenicseeds of the invention are characterized by substantial inhibition ofthe expression of at least one seed storage protein. The secondpreselected DNA sequence is expressed in said transgenic seeds in anamount effective to increase the weight percent of at least one aminoacid present in polypeptide encoded by the second preselected DNAsequence relative to the weight percent of that amino acid innontransgenic seeds. Alternatively, the second preselected DNA sequenceis expressed in transgenic seed in an amount effective to increase theamount, weight percent or level of the polypeptide relative to theamount, weight percent or level of the polypeptide present in a seedtransformed with the second preselected DNA sequence alone.

The invention also provides preselected DNA sequences and expressioncassettes useful in the methods described above, as well as fertiletransgenic plants and/or seeds produced thereby. Preferred fertiletransgenic plants and seeds of the invention exhibit an increase in theweight percent of at least one amino acid essential to the diet ofanimals and/or an increase in the starch content. The fertile transgenicplants and seeds are used to generate true breeding plants so that linesof plants can be developed which transmit the increase in amino acid orstarch content in a dominant fashion while still maintaining thefunctional agronomic characteristics of elite inbred lines. Otherembodiments of the invention include plant cells, plant parts, planttissue and microorganisms transformed with the preselected DNAsequences.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic depicting the functional domains of zein proteins.A consensus amino acid sequence for each of the zein subfamilies isshown. Domains I-IV are shown. Shown in Region IIIb is a consensus ofthe repetitive portion of the zein proteins. Asterisks indicate a lackof consensus at that position. Dots represent gaps inserted to align thesequences.

FIG. 2 is the RNA sequence of A20 (SEQ ID NO:1).

FIG. 3 is the DNA sequence of Z4 (SEQ ID NO:2).

FIG. 4 shows oligonucleotide primers which target the cap site (A) (SEQID NO:9 and SEQ ID NO:10), domain IIIB (B) (SEQ ID NO:11 and SEQ IDNO:12), and the poly(A) region (C) (SEQ ID NO:13 and SEQ ID NO:15) ofthe Z4 gene.

FIG. 5 shows SDS-PAGE analysis of zein extracts from individual kernelsof segregating populations resulting from R1 crosses of a hemizygoustransformant (GW01) carrying pDPG340 and pDPG380 to nontransformedinbreds, and R2 self-pollinations. Lanes 1-8 contain zein extracts fromR2 kernels crossed to CN in the R1 generation and self-pollinated in thesecond generation. Lane 9 contains zein extract from untransformed CN.Lanes 10-17 contain zein extracts from R2 kernels crossed to AW in thefirst generation and self-pollinated in the second generation. Lane 18contains zein extract from untransformed AW. Lane 19 contains molecularweight markers.

FIG. 6 shows SDS-PAGE analysis of zein extracts of vitreous or opaquekernels from segregating populations resulting from crosses ofhemizygous pDPG530 transformants to untransformed inbreds AW and CV.KP014×AW (lanes 1-2); AW×KP014 (lanes 3-4); KP015×AW (lanes 5-6);AW×KP015 (lanes 7-8); CV×KP015 (lanes 9-10); AW×KP015 (lanes 11-12).Lanes 13-19 are AW, CV, ILP, IHP, AK835 opaque, AK835 normal, and W64Aopaque, respectively. Lane 20 contains molecular weight markers.

FIG. 7 shows SDS-PAGE analysis of zein extracts of proteins fromindividual kernels of segregating populations resulting from crosses ofhemizygous transformants and untransformed inbreds. pDPG530 transformantKP015 (AW×KP015, lanes 1-2; CV×KP015, lanes 3-4; KP015×AW, lanes 5-6,and KP016 (CV×KP016, lanes 7-8; KP016×AW, lanes 9-10) and pDPG531transformant KQ018 (KQ018×AW, lanes 11-12). Lanes 13-18 areuntransformed controls CW, AR, CV, AW, W64A, O2 and W64A, respectively.Lanes 19-20 contain molecular weight markers.

FIG. 8 shows α-zein mRNA levels in developing kernels from a segregatingpopulation resulting from crosses of hemizygous pDPG530 and pDPG531transformants to untransformed inbreds AW and CV. AW×KP015 (pDPG530transformant; lanes 1-10; top panel); KP015×AW (pDPG530 transformant;lanes 11-20; top panel); CV×KP015 (pDPG530 transformant; lanes 1-10;lower panel); and KQ012×AW (pDPG531 transformant; lanes 11-20; lowerpanel). Kernels were isolated 21 days post-pollination.

FIG. 9 shows the ultrastructure of pDPG530 transformed (right) anduntransformed (left) kernels.

FIG. 10 shows SDS-PAGE analysis of zein extracts from segregatingpopulations resulting from crosses of pDPG531 transformants tountransformed inbreds AW and CV. CV×KQ012 (lanes 1-4); KQ012×AW (lanes5-8); KQ020×AW (lanes 13-15); KQ020×CV (lanes 16-19). Controls CW, AR,CV and AW (lanes 9-12, respectively). Lane 20 contains a molecularweight marker.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, “substantially identical” or “substantially homologous”in sequence means that two nucleic acid, or amino acid, sequences haveat least about 65%, preferably about 70%, more preferably about 90%, andeven more preferably about 98%, sequence identity, or homology, to eachother. An RNA molecule encoded by a first preselected DNA sequence ofthe invention has sufficient sequence identity or homology to causeco-suppression of the expression of the homologous endogenous gene orexpression of a second preselected DNA sequence which has substantialidentity to the first preselected DNA sequence.

As used herein, “substantially complementary” means that two nucleicacid sequences have at least about 65%, preferably about 70%, morepreferably about 90%, and even more preferably about 98%, sequencecomplementarity to each other. A substantially complementary RNAmolecule is one that has sufficient sequence complementarity to the mRNAencoding a seed storage protein to result in a reduction or inhibitionof the translation of the mRNA.

As used herein, “substantial reduction,” or “substantial decrease” meansthat a transgenic plant, plant part, plant cell or plant tissue has areduced or decreased amount, level or weight percent of a particularamino acid, or polypeptide, relative to the amount, level or weightpercent of that amino acid, or polypeptide, in the correspondingnontransgenic plant, plant part, plant cell or plant tissue. Preferably,the decreased amount, level or weight percent of that amino acid, orpolypeptide, in the transgenic plant, plant part, plant tissue or plantcell is about 10-100% and more preferably about 70%-100%, and even morepreferably about 80-100%, relative to the amount, level or weightpercent of that amino acid, or polypeptide, in the correspondingnontransgenic plant, plant part, plant cell or plant tissue.

As used herein, “increased” or “elevated” levels, amounts or weightpercents of a polypeptide or amino acid in a transformed (transgenic)plant cell, plant tissue, plant part, or plant, are greater than thelevels, amounts or weight percents of that polypeptide or amino acid inthe corresponding untransformed plant cell, plant part, plant tissue, orplant. An increase in the weight percent of an amino acid is an increaseof about 1-50%, preferably about 5-40%, and more preferably about10-30%, in the weight percent of the amino acid in a transgenic plant,plant part, plant tissue, or plant cell relative to the weight percentof that amino acid in a corresponding nontransgenic plant, plant part,plant tissue, or plant cell. An increase in the amount of a polypeptidein a transgenic plant, plant part, plant tissue or plant cell ispreferably at least about 2-100 fold, more preferably at least about3-80 fold, and even more preferably at least about 5-30 fold, relativeto the amount of that polypeptide in the corresponding nontransgenicplant, plant part, plant tissue or plant cell.

For example, the average lysine content in maize seed is about0.24-0.26%, the average methionine content in maize seed is about0.17-0.19%, and the average tryptophan content in maize seed is about0.08-0.10% (Dale, 1996). Thus, the expression of a preselected DNAsequence of the invention in seeds results in an increase in content ofmethionine, tryptophan or lysine in those seeds. The amino acidcomposition of a polypeptide can be determined by methods well known tothe art (Jarrett et al., 1986; Jones et al., 1983; AACC, 1995).

As used herein, “genetically modified” or “transgenic” means a plantcell, plant part, plant tissue or plant which comprises a preselectedDNA segment which is introduced into the genome of a plant cell, plantpart, plant tissue or plant by transformation. The term “wild type”refers to an untransformed plant cell, plant part, plant tissue orplant, i.e., one where the genome has not been altered by the presenceof the preselected DNA segment.

As used herein, “plant” refers to either a whole plant, a plant tissue,a plant part, such as pollen or an embryo, a plant cell, or a group ofplant cells. The class of plants which can be used in the method of theinvention is generally as broad as the class of seed-bearing higherplants amenable to transformation techniques, including bothmonocotyledonous and dicotyledonous plants. Seeds derived from plantsregenerated from transformed plant cells, plant parts or plant tissues,or progeny derived from the regenerated transformed plants, may be useddirectly as feed or food, or can be altered by further processing. Inthe practice of the present invention, the most preferred plant seed isthat of corn or Zea mays. The transformation of the plants in accordancewith the invention may be carried out in essentially any of the variousways known to those skilled in the art of plant molecular biology. Theseinclude, but are not limited to, microprojectile bombardment,microinjection, electroporation of protoplasts or cells comprisingpartial cell walls, and Agrobacterium-mediated DNA transfer.

As used herein, the term “a seed storage protein deficient in at leastone amino acid that is essential to the diet of an animal” means thatthe protein has a lower than average weight percent of at least oneamino acid which is essential to the diet of an animal. Amino acidswhich are essential to the diet of animals include arginine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, threonine,tryptophan and valine. Preferred amino acids which are essential in thediet of animals include methionine, threonine, lysine, isoleucine,tryptophan, and mixtures thereof. A plant seed storage protein cancontain one or more of these essential amino acids. For example, theaverage weight percent of lysine in a maize seed is about about0.24-0.26%. Thus, a seed storage protein, such as an α-zein, which doesnot comprise lysine, is deficient in lysine. The average weight percentof a particular amino acid is determined by methods well known to theart.

As used herein, “isolated” means either physically isolated from thecell or synthesized in vitro in the basis of the sequence of an isolatedDNA segment.

As used herein, a “native” gene means a DNA sequence or segment that hasnot been manipulated in vitro, i.e., has not been isolated, purified,and amplified.

I. DNA Molecules of the Invention

A. Isolation of Preselected Sense and Antisense DNA Sequences

1. α-Zein Seed Storage Proteins

A genetically engineered, isolated purified DNA molecule useful in theinvention can comprise a preselected DNA sequence encoding an RNAmolecule substantially homologous, or complementary, to all or a portionthereof of a mRNA coding for a plant seed storage protein, e.g., one ofthe α-zein proteins. As used herein, a “seed storage protein” is aprotein which is one of the major proteins in mature seeds of plantssuch as maize, and comprises a signal peptide sequence at the aminoterminal end of the pre-form of the protein, and which comprises atandem repeat of amino acid sequences in the mature form of the protein.

Plant seed storage proteins or zein proteins include, but are notlimited to, zein proteins, such as α-zeins, e.g., proteins of 19,000 and22,000 daltons; β-zein proteins, e.g., proteins with a molecular weightof 14,000 daltons, γ-zein proteins, e.g., proteins with molecularweights of 27,000 and 16,000 daltons; and δ-zein proteins, i.e.,proteins with a molecular weight of 10,000 daltons. Certain seed storageproteins are deficient in at least one amino acid essential to the dietof animals. For example, the 19 kD and 22 kD α-zein proteins contain lowlevels of the amino acids lysine and tryptophan which are essential tothe diet of animals.

In an alternative embodiment, the preselected DNA sequence is expressedas a RNA molecule that is substantially complementary to, or identicalto, respectively, all or a portion of a family-, or subfamily-, of seedstorage protein specific mRNA. The RNA molecule, or corresponding DNAsequence, has about 65%, or more preferably 90%, nucleic acid sequencehomology or complementarity with other RNA, or DNA, respectively,sequences which encode seed storage proteins of the same family orsubfamily. The expression of a preselected antisense DNA sequencesubstantially inhibits translation of the complementary mRNA, while theexpression of a preselected sense DNA sequence results in cosuppressionof the expression of endogenous DNA sequences encoding the homologousseed storage proteins. A preferred preselected DNA molecule encodes anRNA molecule which is complementary to the DNA sequence which encodesthe tandem repeat region of 20 amino acids of the same family orsubfamily of seed storage proteins.

The preselected sense or antisense DNA sequence can encode an RNAmolecule preferably having about 15 nucleotides to 2,000 nucleotides andmore preferably about 50-1,000 nucleotides. The DNA sequence can bederived from the 5′ terminus or the 3′ terminus and can include all oronly a portion of the coding and/or noncoding regions. It will beunderstood by those of skill in the art that a sense or antisense DNAsequence should provide an RNA sequence having at least about 15nucleotides in order to provide for substantial inhibition of theexpression of the mRNA coding for the seed storage protein.

The preselected DNA sequences of the invention are obtained by cloning aDNA molecule, sequence or segment which encodes, and can be expressed asa mRNA of, a seed storage protein. Portions of the preselected DNAsequence can also include noncoding nucleotides located at either the 5′or 3′ ends of the sense coding sequence. A preselected DNA sequencewhich encodes an RNA sequence that is substantially complementary to amRNA sequence encoding a seed storage protein is typically a “sense” DNAsequence cloned in the opposite orientation (i.e., 3′ to 5′ rather than5′ to 3′). A sense DNA sequence encoding a seed storage protein can becloned using standard methods as described in Sambrook et al. (1989),and U.S. Pat. No. 5,508,468.

A subfragment of a preselected DNA sequence which encodes a full-lengthseed storage protein can be generated using restriction enzymes. Thesubfragment is preferably selected based upon the known functionaldomains of seed storage proteins. A seed storage protein has at leastfour different functional domains: a signal peptide domain, a domainwhich includes the amino terminal portion of the mature protein which islocated downstream of the signal peptide, a domain which includes tandemrepeats of a 20 amino acid sequence which is located downstream of theamino terminus of the mature protein, and a domain which includes thecarboxy terminus of the protein. The size and location of thesefunctional domains in the α-zein proteins are shown in FIG. 1 and can bedetermined for other seed storage proteins by comparing the amino acidsequence of other seed storage proteins to the amino acid sequence ofthe α-zein proteins.

Suitable examples of preselected DNA sequences that can provide all or aportion of a sense or antisense seed storage protein, e.g., α-zein, DNAsequence include cDNA clones A20, A30, B49, B59, B36, Z4, and Z15prepared as described by Messing et al. (1983). Preferred cDNA clonesare an A20 clone, which encodes a 19 kD α-zein protein, and a Z4 clone,which encodes a 22 kD α-zein protein. Portions of the Z4 and the A20 DNAsequences can be generated with restriction endonucleases.

It is also contemplated that preselected DNA sequences homologous orcomplementary to any portion of the A20 or Z4 RNA, in vectorsappropriate for expression in plants, may be used to substantiallydecrease the production of seed storage proteins. Examples of such DNAsequences are sequences which may be homologous or complementary to the5′ region of the DNA or RNA sequence such as the 3′ region of thepromoter and the cap site (FIG. 4A), or the 3′ region of the gene suchas the AATAAA-like polyadenylation signal, upstream of the poly(A) tail(FIG. 4C). It is further contemplated that a preselected DNA sequencehomologous or complementary to a conserved domain common to more thanone gene in a gene family or subfamily, such as domain IIIB or one ormore of the other domains shown in FIG. 1, may also be useful tosubstantially inhibit the expression of members of the gene family orsubfamily (FIG. 4B). It is further contemplated that the preselected DNAsequence may encode an RNA molecule which is substantially identical toall or a portion of a mRNA encoding a seed storage protein, e.g., apreselected DNA sequence encoding a RNA molecule substantially identicalto the mRNA encoding 10 kD zein, 27 kD zein, or MB1.

In a preferred embodiment, a sense DNA sequence encoding a 19 kD α-zeinprotein and/or a sense DNA sequence encoding a 22 kD α-zein protein isprepared from a cDNA library generated from endosperm tissue asdescribed in Hu et al. (1982) and Geraghty et al. (1982), which arehereby incorporated by reference. The cDNA clones encoding a 19 kDα-zein protein and/or a 22 kD α-zein protein can be characterized bystandard methods such as DNA hybridization or detection of geneexpression by immunotechniques including Western blot analysis. Thepresence of the coding sequence of the 19 kD or 22 kD α-zein protein canbe confirmed by DNA sequencing.

2. Other Preselected DNA Sentences

Another preselected DNA sequence useful in the method of the inventionencodes a polypeptide, including a plant protein, comprising at leastone amino acid essential to the diet of animals operably linked to apromoter functional in a plant and/or seeds. The expression of thepreselected DNA sequence, coding for the polypeptide comprising at leastone amino acid essential to the diet of animals, in a plant cellprovides for an increase in expression of the polypeptide so that theweight percent of the amino acid residue is substantially increased inthe plant regenerated from the transformed plant cell, or seed derivedfrom said plant, over the amount normally present in the correspondinguntransformed plant or seed. Preferably, the preselected DNA sequence isco-transformed into plant cells with a second preselected antisense orsense DNA sequence, the expression of which results in the inhibition ofexpression of a seed storage protein relatively deficient in an aminoacid essential in the diet of animals.

The preselected DNA sequence coding for a polypeptide comprising atleast one amino acid essential in the diet of animals may be apolypeptide expressed in a plant seed, such as a 10 kD zein protein.Other polypeptides that contain one or more amino acid residuesessential in the diet of animals include the synthetic polypeptide MB1(Beauregard et al., 1995). It is contemplated that any gene encoding anaturally occurring polypeptide, or a synthetic polypeptide, thatcontains at least one amino acid essential in the diet of an animal maybe used in the present invention. The Z10 and MB1 proteins areillustrative of a naturally occurring protein and a syntheticpolypeptide, respectively, although one of skill in the art will realizethat many other proteins are useful in the practice of the presentinvention.

The preselected DNA sequences encoding these polypeptides can beobtained by standard methods, as described by Sambrook et al., citedsupra. For example, a cDNA clone encoding a 10 kD zein protein can beobtained from maize endosperm tissue, as described by Kirihara et al.(1988). The DNA sequence is then preferably combined with a promoterthat is functional in plant cells or seeds. The preferred promoter is apromoter functional during plant seed development, such as the Z27 orZ10 promoter.

The gene encoding the synthetic polypeptide MB1 is obtained from Mary A.Hefford (Center for Food and Animal Research, Agriculture and Agri-FoodCanada). The preselected DNA sequence encoding a synthetic polypeptidesuch as MB1 is operably linked to a signal sequence derived from a seedstorage protein. For example, the MB1 DNA sequence can be operablylinked to the 15 kD zein signal peptide sequence.

It is also contemplated that a preselected DNA sequence encodes adesirable seed storage protein. Thus, the expression of a firstpreselected DNA sequence can inhibit the expression of an undesirableseed storage protein, while the expression of a second preselected DNAsequence can encode a desirable gene product, e.g., a desirable seedstorage protein. For example, it is envisioned that the expression ofthe first preselected DNA sequence, which comprises partial gene DNAsequences, may be advantageous for the suppression of the expression ofundesirable seed storage proteins, if those partial DNA sequences targetDNA or RNA sequences not present in the second preselected DNA sequencewhich encodes a desirable polypeptide, e.g., 10 kD zein or MB1, in orderto avoid suppression of expression of the desirable polypeptide.

B. Optional Sequences for Expression Cassettes

1. Promoters

Preferably, the preselected DNA sequence of the invention is operablylinked to a promoter, which provides for expression of the preselectedDNA sequence. The promoter is preferably a promoter functional in plantsand/or seeds, and more preferably a promoter functional during plantseed development. A preselected DNA sequence is operably linked to thepromoter when it is located downstream from the promoter, to form anexpression cassette.

Most endogenous genes have regions of DNA that are known as promoters,which regulate gene expression. Promoter regions are typically found inthe flanking DNA upstream from the coding sequence in both prokaryoticand eukaryotic cells. A promoter sequence provides for regulation oftranscription of the downstream gene sequence and typically includesfrom about 50 to about 2,000 nucleotide base pairs. Promoter sequencesalso contain regulatory sequences such as enhancer sequences that caninfluence the level of gene expression. Some isolated promoter sequencescan provide for gene expression of heterologous DNAs, that is a DNAdifferent from the native or homologous DNA.

Promoter sequences are also known to be strong or weak, or inducible. Astrong promoter provides for a high level of gene expression, whereas aweak promoter provides for a very low level of gene expression. Aninducible promoter is a promoter that provides for the turning on andoff of gene expression in response to an exogenously added agent, or toan environmental or developmental stimulus. A bacterial promoter such asthe P_(tac) promoter can be induced to varying levels of gene expressiondepending on the level of isothiopropylgalactoside added to thetransformed bacterial cells. Promoters can also provide for tissuespecific or developmental regulation. An isolated promoter sequence thatis a strong promoter for heterologous DNAs is advantageous because itprovides for a sufficient level of gene expression to allow for easydetection and selection of transformed cells and provides for a highlevel of gene expression when desired.

Preferred expression cassettes will generally include, but are notlimited to, a plant promoter such as the CaMV 35S promoter (Odell etal., 1985), or others such as CaMV 19S (Lawton et al., 1987), nos (Ebertet al., 1987), Adh1 (Walker et al., 1987), sucrose synthase (Yang etal., 1990), α-tubulin, ubiquitin, actin (Wang et al., 1992), cab(Sullivan et al., 1989), PEPCase (Hudspeth et al., 1989) or thoseassociated with the R gene complex (Chandler et al., 1989). Furthersuitable promoters include cauliflower mosaic virus promoter, the Z10promoter from a gene encoding a 10 kD zein protein, a Z27 promoter froma gene encoding a 27 kD zein protein, inducible promoters, such as thelight inducible promoter derived from the pea rbcS gene (Coruzzi et al.,1971) and the actin promoter from rice (McElroy et al., 1990); seedspecific promoters, such as the phaseolin promoter from beans, may alsobe used (Sengupta-Gopalan, 1985). The especially preferred promoter isfunctional during plant seed development, such as the Z10 or Z27promoters. Other promoters useful in the practice of the invention areknown to those of skill in the art.

Alternatively, novel tissue-specific promoter sequences may be employedin the practice of the present invention. cDNA clones from a particulartissue are isolated and those clones which are expressed specifically inthat tissue are identified, for example, using Northern blotting.Preferably, the gene isolated is not present in a high copy number, butis relatively abundant in specific tissues. The promoter and controlelements of corresponding genomic clones can then be localized usingtechniques well known to those of skill in the art.

A preselected DNA sequence can be combined with the promoter by standardmethods as described in Sambrook et al., cited supra, to yield anexpression cassette. Briefly, a plasmid containing a promoter such asthe 35S CaMV promoter can be constructed as described in Jefferson(1987) or obtained from Clontech Lab in Palo Alto, Calif. (e.g., pBI121or pBI221). Typically, these plasmids are constructed to have multiplecloning sites having specificity for different restriction enzymesdownstream from the promoter. The preselected DNA sequence can besubcloned downstream from the promoter using restriction enzymes andpositioned to ensure that the DNA is inserted in proper orientation withrespect to the promoter so that the DNA can be expressed as sense orantisense RNA. Once the preselected DNA sequence is operably linked to apromoter, the expression cassette so formed can be subcloned into aplasmid or other vector.

Once the preselected sense DNA sequence is obtained, all or a portion ofthe DNA sequence can be subcloned into an expression vector (see below)in the opposite orientation (i.e., 3′ to 5′). Similarly, all or aportion of the preselected DNA sequence can be subcloned in senseorientation (i.e., 5′ to 3′). The preselected DNA sequence is subcloneddownstream from a promoter to form an expression cassette.

In a preferred embodiment, a cDNA clone encoding a Z4 22 kD α-zeinprotein is isolated from maize endosperm tissue. Using restrictionendonucleases, the entire coding sequence for the Z4 gene is subclonedin the 3′ to 5′ orientation into an intermediate vector to form anantisense DNA sequence. The promoter region from a 10 kD zein protein,designated the Z10 promoter, is subcloned upstream from the antisenseDNA sequence which includes the entire coding sequence for the Z4 geneto form an expression cassette. This expression cassette can then besubcloned into a vector suitable for transformation of plant cells. Inanother preferred embodiment of the present invention, the promoterregion from a 27 kD zein protein, designated the Z27 promoter, issubcloned upstream from the antisense DNA sequence.

In another preferred embodiment of the present invention, usingrestriction endonucleases, the entire coding sequence of the A20 geneencoding a 19 kD α-zein protein is subcloned in the 3′ to 5′ orientationinto an intermediate vector to form an antisense DNA sequence. The Z10promoter, or alternatively the Z27 promoter, is cloned upstream from theA20 antisense DNA sequence. Partial Z4 or A20 DNA sequences can also becloned in an antisense 3′ to 5′ orientation downstream of the Z10 or Z27promoter. Furthermore, it is contemplated that expression cassettes maybe constructed which comprise the Z10 or Z27 promoter upstream of apartial or entire Z4 or A20 DNA sequences wherein said DNA sequences aresubcloned downstream of the promoter in a 5′ to 3′ sense orientation.

2. Targeting Sequences

Additionally, expression cassettes can be constructed and employed totarget the product of the preselected DNA sequence or segment to anintracellular compartment within plant cells or to direct a protein tothe extracellular environment. This can generally be achieved by joininga DNA sequence encoding a transit or signal peptide sequence to thecoding sequence of the preselected DNA sequence. The resultant transit,or signal, peptide will transport the protein to a particularintracellular, or extracellular destination, respectively, and can thenbe post-translationally removed. Transit peptides act by facilitatingthe transport of proteins through intracellular membranes, e.g.,vacuole, vesicle, plastid and mitochondrial membranes, whereas signalpeptides direct proteins through the extracellular membrane. Byfacilitating transport of the protein into compartments inside oroutside the cell, these sequences can increase the accumulation of aparticular gene product in a particular location. For example, see U.S.Pat. No. 5,258,300.

3. 3′ Sequences

When the expression cassette is to be introduced into a plant cell, theexpression cassette can also optionally include 3′ nontranslated plantregulatory DNA sequences that act as a signal to terminate transcriptionand allow for the polyadenylation of the resultant mRNA. The 3′nontranslated regulatory DNA sequence preferably includes from about 300to 1,000 nucleotide base pairs and contains plant transcriptional andtranslational termination sequences. Preferred 3′ elements are derivedfrom those from the nopaline synthase gene of Agrobacterium tumefaciens(Bevan et al., 1983), the terminator for the T7 transcript from theoctopine synthase gene of Agrobacterium tumefaciens, and the 3′ end ofthe protease inhibitor I or II genes from potato or tomato, althoughother 3′ elements known to those of skill in the art can also beemployed. These 3′ nontranslated regulatory sequences can be obtained asdescribed in An (1987) or are already present in plasmids available fromcommercial sources such as Clontech, Palo Alto, Calif. The 3′nontranslated regulatory sequences can be operably linked to the 3′terminus of the preselected DNA sequence by standard methods.

4. Selectable and Screenable Marker Sequences

In order to improve the ability to identify transformants, one maydesire to employ a selectable or screenable marker gene as, or inaddition to, the expressible preselected DNA sequence or segment.“Marker genes” are genes that impart a distinct phenotype to cellsexpressing the marker gene and thus allow such transformed cells to bedistinguished from cells that do not have the marker. Such genes mayencode either a selectable or screenable marker, depending on whetherthe marker confers a trait which one can ‘select’ for by chemical means,i.e., through the use of a selective agent (e.g., a herbicide,antibiotic, or the like), or whether it is simply a trait that one canidentify through observation or testing, i.e., by ‘screening’ (e.g., theR-locus trait). Of course, many examples of suitable marker genes areknown to the art and can be employed in the practice of the invention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or even secretable enzymes which can bedetected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA; and proteins that are inserted or trapped in the cellwall (e.g., proteins that include a leader sequence such as that foundin the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a polypeptide that becomes sequestered in the cell wall, andwhich polypeptide includes a unique epitope is considered to beparticularly advantageous. Such a secreted antigen marker would ideallyemploy an epitope sequence that would provide low background in planttissue, a promoter-leader sequence that would impart efficientexpression and targeting across the plasma membrane, and would produceprotein that is bound in the cell wall and yet accessible to antibodies.A normally secreted wall protein modified to include a unique epitopewould satisfy all such requirements.

One example of a protein suitable for modification in this manner isextensin, or hydroxyproline rich glycoprotein (HPRG). The use of themaize HPRG (Stiefel et al., 1990) is preferred as this molecule is wellcharacterized in terms of molecular biology, expression, and proteinstructure. However, any one of a variety of extensins and/orglycine-rich wall proteins (Keller et al., 1989) could be modified bythe addition of an antigenic site to create a screenable marker.

Elements of the present disclosure are exemplified in detail through theuse of particular marker genes. However in light of this disclosure,numerous other possible selectable and/or screenable marker genes willbe apparent to those of skill in the art in addition to the one setforth herein below. Therefore, it will be understood that the followingdiscussion is exemplary rather than exhaustive. In light of thetechniques disclosed herein and the general recombinant techniques whichare known in the art, the present invention renders possible theintroduction of any gene, including marker genes, into a recipient cellto generate a transformed plant cell, e.g., a monocot cell.

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, a neo gene (Potrykus et al.,1985) which codes for kanamycin resistance and can be selected for usingkanamycin, G418, and the like; a bar gene which codes for bialaphosresistance; a gene which encodes an altered EPSP synthase protein(Hinchee et al., 1988) thus conferring glyphosate resistance; anitrilase gene such as bxn from Klebsiella ozaenae which confersresistance to bromoxynil (Stalker et al., 1988); a mutant acetolactatesynthase gene (ALS) which confers resistance to imidazolinone,sulfonylurea or other ALS-inhibiting chemicals (European PatentApplication 154,204, 1985); a methotrexate-resistant DHFR gene (Thilletet al., 1988); a dalapon dehalogenase gene that confers resistance tothe herbicide dalapon; or a mutated anthranilate synthase gene thatconfers resistance to 5-methyl tryptophan. Where a mutant EPSP synthasegene is employed, additional benefit may be realized through theincorporation of a suitable chloroplast transit peptide, CTP (EuropeanPatent Application 0 218 571, 1987).

An illustrative embodiment of a selectable marker gene capable of beingused in systems to select transformants is the genes that encode theenzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes (U.S. Pat. No. 5,550,318, which is incorporated byreference herein). The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., 1986; Twell et al., 1989) causing rapid accumulation of ammonia andcell death. The success in using this selective system in conjunctionwith monocots was particularly surprising because of the majordifficulties which have been reported in transformation of cereals(Potrykus, 1989).

Screenable markers that may be employed include, but are not limited to,a β-glucuronidase or uidA gene (GUS) which encodes an enzyme for whichvarious chromogenic substrates are known; an R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., 1988); a β-lactamase gene(Sutcliffe, 1978), which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); a xyIEgene (Zukowsky et al., 1983) which encodes a catechol dioxygenase thatcan convert chromogenic catechols; an α-amylase gene (Ikuta et al.,1990); a tyrosinase gene (Katz et al., 1983) which encodes an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone which in turncondenses to form the easily detectable compound melanin; aβ-galactosidase gene, which encodes an enzyme for which there arechromogenic substrates; a luciferase (lux) gene (Ow et al., 1986), whichallows for bioluminescence detection; or an aequorin gene (Prasher etal., 1985), which may be employed in calcium-sensitive bioluminescencedetection, or a green fluorescent protein gene (Niedz et al., 1995).

Genes from the maize R gene complex are contemplated to be particularlyuseful as screenable markers. The R gene complex in maize encodes aprotein that acts to regulate the production of anthocyanin pigments inmost seed and plant tissue. Maize strains can have one, or as many asfour, R alleles which combine to regulate pigmentation in adevelopmental and tissue specific manner. A gene from the R gene complexwas applied to maize transformation, because the expression of this genein transformed cells does not harm the cells. Thus, an R gene introducedinto such cells will cause the expression of a red pigment and, ifstably incorporated, can be visually scored as a red sector. If a maizeline carries dominant alleles for genes encoding the enzymaticintermediates in the anthocyanin biosynthetic pathway (C2, A1, A2, Bz1and Bz2), but carries a recessive allele at the R locus, transformationof any cell from that line with R will result in red pigment formation.Exemplary lines include Wisconsin 22 which contains the rg-Stadlerallele and TR112, a K55 derivative which is r-g, b, P1. Alternativelyany genotype of maize can be utilized if the C1 and R alleles areintroduced together.

It is further proposed that R gene regulatory regions may be employed inchimeric constructs in order to provide mechanisms for controlling theexpression of chimeric genes. More diversity of phenotypic expression isknown at the R locus than at any other locus (Coe et al., 1988). It iscontemplated that regulatory regions obtained from regions 5′ to thestructural R gene would be valuable in directing the expression ofgenes, e.g., insect resistance, drought resistance, herbicide toleranceor other protein coding regions. For the purposes of the presentinvention, it is believed that any of the various R gene family membersmay be successfully employed (e.g., P, S, Lc, etc.). However, the mostpreferred will generally be Sn (particularly Sn:bol3). Sn is a dominantmember of the R gene complex and is functionally similar to the R and Bloci in that Sn controls the tissue specific deposition of anthocyaninpigments in certain seedling and plant cells, therefore, its phenotypeis similar to R.

A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulational screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening.

5. Other Optional Sequences

An expression cassette of the invention can also further compriseplasmid DNA. Plasmid vectors include additional DNA sequences thatprovide for easy selection, amplification, and transformation of theexpression cassette in prokaryotic and eukaryotic cells, e.g.,pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUC119, andpUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors,or pBS-derived vectors. The additional DNA sequences include origins ofreplication to provide for autonomous replication of the vector,additional selectable marker genes, preferably encoding antibiotic orherbicide resistance, unique multiple cloning sites providing formultiple sites to insert DNA sequences or genes encoded in theexpression cassette, and sequences that enhance transformation ofprokaryotic and eukaryotic cells.

Another vector that is useful for expression in both plant andprokaryotic cells is the binary Ti plasmid (as disclosed in Schilperoortet al., U.S. Pat. No. 4,940,838) as exemplified by vector pGA582. Thisbinary Ti plasmid vector has been previously characterized by An, citedsupra, and is available from Dr. An. This binary Ti vector can bereplicated in prokaryotic bacteria such as E. coli and Agrobacterium.The Agrobacterium plasmid vectors can be used to transfer the expressioncassette to dicot plant cells, and under certain conditions to monocotcells, such as rice cells. The binary Ti vectors preferably include thenopaline T DNA right and left borders to provide for efficient plantcell transformation, a selectable marker gene, unique multiple cloningsites in the T border regions, the colE1 replication of origin and awide host range replicon. The binary Ti vectors carrying an expressioncassette of the invention can be used to transform both prokaryotic andeukaryotic cells, but is preferably used to transform dicot plant cells.

C. In Vitro Screening of Expression Cassettes

Once the expression cassette is constructed and subcloned into asuitable plasmid, it can be screened for the ability to substantiallyinhibit the translation of a mRNA coding for a seed storage protein bystandard methods such as hybrid arrested translation. For example, forhybrid selection or arrested translation, a preselected antisense DNAsequence is subcloned into an SP6/T7 containing plasmids (as supplied byProMega Corp.). For transformation of plants cells, suitable vectorsinclude plasmids such as described herein. Typically, hybrid arresttranslation is an in vitro assay which measures the inhibition oftranslation of a mRNA encoding a particular seed storage protein. Thisscreening method can also be used to select and identify preselectedantisense DNA sequences that inhibit translation of a family orsubfamily of zein protein genes. As a control, the corresponding senseexpression cassette is introduced into plants and the phenotype assayed.

II. DNA Delivery of the DNA Molecules into Host Cells

The present invention generally includes steps directed to introducing apreselected DNA sequence, such as a preselected cDNA, into a recipientcell to create a transformed cell. The frequency of occurrence of cellstaking up exogenous (foreign) DNA is believed to be low. Moreover, it ismost likely that not all recipient cells receiving DNA segments orsequences will result in a transformed cell wherein the DNA is stablyintegrated into the plant genome and/or expressed. Some may show onlyinitial and transient gene expression. However, certain cells fromvirtually any dicot or monocot species may be stably transformed, andthese cells regenerated into transgenic plants, through the applicationof the techniques disclosed herein.

The invention is directed to any plant species wherein the seed containsstorage proteins that contain relatively low levels, or none, of atleast one essential amino acid. Cells of the plant tissue source arepreferably embryogenic cells or cell-lines that can regenerate fertiletransgenic plants and/or seeds. The cells can be derived from eithermonocotyledons or dicotyledons. Suitable examples of plant speciesinclude wheat, rice, Arabidopsis, tobacco, maize, soybean, and the like.The preferred cell type is a monocotyledon cell such as a maize cell,which may be in a suspension cell culture or may be in an intact plantpart, such as an immature embryo, or in a specialized plant tissue, suchas callus, such as Type I or Type II callus.

Transformation of the cells of the plant tissue source can be conductedby any one of a number of methods known to those of skill in the art.Examples are: Transformation by direct DNA transfer into plant cells byelectroporation (U.S. Pat. No. 5,384,253 and U.S. Pat. No. 5,472,869,incorporated herein by reference; Dekeyser et al., 1990); direct DNAtransfer to plant cells by PEG precipitation (Hayashimoto et al., 1990);direct DNA transfer to plant cells by microprojectile bombardment(McCabe et al., 1988; Gordon-Kamm et al., 1990; U.S. Pat. No. 5,489,520;U.S. Pat. No. 5,538,877; and U.S. Pat. No. 5,538,880, incorporatedherein by reference) and DNA transfer to plant cells via infection withAgrobacterium. Methods such as microprojectile bombardment orelectroporation can be carried out with “naked” DNA where the expressioncassette may be simply carried on any E. coli-derived plasmid cloningvector. In the case of viral vectors, it is desirable that the systemretain replication functions, but lack functions for disease induction.

The preferred method for dicot transformation is via infection of plantcells with Agrobacterium tumefaciens using the leaf-disk protocol(Horsch et al., 1985). Monocots such as Zea mays can be transformed viamicroprojectile bombardment of embryogenic callus tissue or immatureembryos, or by electroporation following partial enzymatic degradationof the cell wall with a pectinase-containing enzyme (U.S. Pat. No.5,384,253; and U.S. Pat. No. 5,472,869). For example, embryogenic celllines derived from immature Zea mays embryos can be transformed byaccelerated particle treatment as described by Gordon-Kamm et al. (1990)or U.S. Pat. No. 5,489,520; U.S. Pat. No. 5,538,877 and U.S. Pat. No.5,538,880, cited above. Excised immature embryos can also be used as thetarget for transformation prior to tissue culture induction, selectionand regeneration as described in U.S. application Ser. No. 08/112,245and PCT publication WO 95/06128. Furthermore, methods for transformationof monocotyledonous plants utilizing Agrobacterium tumefaciens have beendescribed by Hiei et al. (European Patent 0 604 662, 1994) and Saito etal. (European Patent 0 672 752, 1995).

Methods such as microprojectile bombardment or electroporation arecarried out with “naked” DNA where the expression cassette may be simplycarried on any E. coli-derived plasmid cloning vector. In the case ofviral vectors, it is desirable that the system retain replicationfunctions, but lack functions for disease induction.

The choice of plant tissue source for transformation will depend on thenature of the host plant and the transformation protocol. Useful tissuesources include callus, suspension culture cells, protoplasts, leafsegments, stem segments, tassels, pollen, embryos, hypocotyls, tubersegments, meristematic regions, and the like. The tissue source isselected and transformed so that it retains the ability to regeneratewhole, fertile plants following transformation, i.e., containstotipotent cells. Type I or Type II embryonic maize callus and immatureembryos are preferred Zea mays tissue sources. Selection of tissuesources for transformation of monocots is described in detail in U.S.Application Serial No. 08/112,245 and PCT publication WO 95/06128(incorporated herein by reference).

The transformation is carried out under conditions directed to the planttissue of choice. The plant cells or tissue are exposed to the DNAcarrying the preselected DNA sequences for an effective period of time.This may range from a less-than-one-second pulse of electricity forelectroporation to a 2-3 day co-cultivation in the presence ofplasmid-bearing Agrobacterium cells. Buffers and media used will alsovary with the plant tissue source and transformation protocol. Manytransformation protocols employ a feeder layer of suspended culturecells (tobacco or Black Mexican Sweet corn, for example) on the surfaceof solid media plates, separated by a sterile filter paper disk from theplant cells or tissues being transformed.

A. Electroporation

Where one wishes to introduce DNA by means of electroporation, it iscontemplated that the method of Krzyzek et al (U.S. Pat. No. 5,384,253,incorporated herein by reference) will be particularly advantageous. Inthis method, certain cell wall-degrading enzymes, such aspectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells. Alternatively, recipient cells can be made moresusceptible to transformation, by mechanical wounding.

To effect transformation by electroporation, one may employ eitherfriable tissues such as a suspension cell cultures, or embryogeniccallus, or alternatively, one may transform immature embryos or otherorganized tissues directly. The cell walls of the preselected cells ororgans can be partially degraded by exposing them to pectin-degradingenzymes (pectinases or pectolyases) or mechanically wounding them in acontrolled manner. Such cells would then be receptive to DNA uptake byelectroporation, which may be carried out at this stage, and transformedcells then identified by a suitable selection or screening protocoldependent on the nature of the newly incorporated DNA.

B. Microprojectile Bombardment

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method,microparticles may be coated with DNA and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

It is contemplated that in some instances DNA precipitation onto metalparticles would not be necessary for DNA delivery to a recipient cellusing microprojectile bombardment. In an illustrative embodiment,non-embryogenic BMS cells were bombarded with intact cells of thebacteria E. coli or Agrobacterium tumefaciens containing plasmids witheither the β-glucoronidase or bar gene engineered for expression inmaize. Bacteria were inactivated by ethanol dehydration prior tobombardment. A low level of transient expression of the β-glucoronidasegene was observed 24-48 hours following DNA delivery. In addition,stable transformants containing the bar gene were recovered followingbombardment with either E. coli or Agrobacterium tumefaciens cells. Itis contemplated that particles may contain DNA rather than be coatedwith DNA. Hence it is proposed that particles may increase the level ofDNA delivery but are not, in and of themselves, necessary to introduceDNA into plant cells.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming monocots, is thatthe isolation of protoplasts (Christou et al., 1988), the formation ofpartially degraded cells, or the susceptibility to Agrobacteriuminfection is required. An illustrative embodiment of a method fordelivering DNA into maize cells by acceleration is a Biolistics ParticleDelivery System, which can be used to propel particles coated with DNAor cells through a screen, such as a stainless steel or Nytex screen,onto a filter surface covered with maize cells cultured in suspension(Gordon-Kamm et al., 1990). The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectile aggregate andmay contribute to a higher frequency of transformation, by reducingdamage inflicted on the recipient cells by an aggregated projectile.

For bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein one may obtainup to 1000 or more foci of cells transiently expressing a marker gene.The number of cells in a focus which express the exogenous gene product48 hours post-bombardment often range from about 1 to 10 and averageabout 1 to 3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the path and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmidDNA. It is believed that prebombardment manipulations are especiallyimportant for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance, and helium pressure.One may also minimize the trauma reduction factors (TRFs) by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells may be adjustedfor optimum transformation. Results from such small scale optimizationstudies are disclosed herein and the execution of other routineadjustments will be known to those of skill in the art in light of thepresent disclosure.

III. Production and Characterization of Stable Transgenic Maize

After effecting delivery of a preselected DNA sequence to recipientcells by any of the methods discussed above, the next steps of theinvention generally concern identifying the transformed cells forfurther culturing and plant regeneration. As mentioned above, in orderto improve the ability to identify transformants, one may desire toemploy a selectable or screenable marker gene as, or in addition to, theexpressible preselected DNA sequence. In this case, one would thengenerally assay the potentially transformed cell population by exposingthe cells to a selective agent or agents, or one would screen the cellsfor the desired marker gene trait.

A. Selection

An exemplary embodiment of methods for identifying transformed cellsinvolves exposing the bombarded cultures to a selective agent, such as ametabolic inhibitor, an antibiotic, herbicide or the like. Cells whichhave been transformed and have stably integrated a marker geneconferring resistance to the selective agent used, will grow and dividein culture. Sensitive cells will not be amenable to further culturing.

To use the bar-bialaphos or the EPSPS-glyphosate selective system,bombarded tissue is cultured for about 0-28 days on nonselective mediumand subsequently transferred to medium containing from about 1-3 mg/lbialaphos or about 1-3 mM glyphosate, as appropriate. While ranges ofabout 1-3 mg/l bialaphos or about 1-3 mM glyphosate will typically bepreferred, it is proposed that ranges of at least about 0.1-50 mg/lbialaphos or at least about 0.1-50 mM glyphosate will find utility inthe practice of the invention. Tissue can be placed on any porous,inert, solid or semi-solid support for bombardment, including but notlimited to filters and solid culture medium. Bialaphos and glyphosateare provided as examples of agents suitable for selection oftransformants, but the technique of this invention is not limited tothem.

An example of a screenable marker trait is the red pigment producedunder the control of the R-locus in maize. This pigment may be detectedby culturing cells on a solid support containing nutrient media capableof supporting growth at this stage and selecting cells from colonies(visible aggregates of cells) that are pigmented. These cells may becultured further, either in suspension or on solid media. The R-locus isuseful for selection of transformants from bombarded immature embryos.In a similar fashion, the introduction of the C1 and B genes will resultin pigmented cells and/or tissues.

The enzyme luciferase is also useful as a screenable marker in thecontext of the present invention. In the presence of the substrateluciferin, cells expressing luciferase emit light which can be detectedon photographic or x-ray film, in a luminometer (or liquid scintillationcounter), by devices that enhance night vision, or by a highly lightsensitive video camera, such as a photon counting camera. All of theseassays are nondestructive and transformed cells may be cultured furtherfollowing identification. The photon counting camera is especiallyvaluable as it allows one to identify specific cells or groups of cellswhich are expressing luciferase and manipulate those in real time.

It is further contemplated that combinations of screenable andselectable markers will be useful for identification of transformedcells. In some cell or tissue types a selection agent, such as bialaphosor glyphosate, may either not provide enough killing activity to clearlyrecognize transformed cells or may cause substantial nonselectiveinhibition of transformants and nontransformants alike, thus causing theselection technique to not be effective. It is proposed that selectionwith a growth inhibiting compound, such as bialaphos or glyphosate atconcentrations below those that cause 100% inhibition followed byscreening of growing tissue for expression of a screenable marker genesuch as luciferase would allow one to recover transformants from cell ortissue types that are not amenable to selection alone. In anillustrative embodiment embryogenic Type II callus of Zea mays L. wasselected with sub-lethal levels of bialaphos. Slowly growing tissue wassubsequently screened for expression of the luciferase gene andtransformants were identified. In this example, neither selection norscreening conditions employed were sufficient in and of themselves toidentify transformants. Therefore it is proposed that combinations ofselection and screening will enable one to identify transformants in awider variety of cell and tissue types.

B. Regeneration and Seed Production

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In an exemplary embodiment, MS andN6 media have been modified (see Table 1 of U.S. application Ser. No.08/594,861, the disclosure of which is incorporated by reference herein)by including further substances such as growth regulators. A preferredgrowth regulator for such purposes is dicamba or 2,4-D. However, othergrowth regulators may be employed, including NAA, NAA+2,4-D or perhapseven picloram. Media improvement in these and like ways was found tofacilitate the growth of cells at specific developmental stages. Tissueis preferably maintained on a basic media with growth regulators untilsufficient tissue is available to begin plant regeneration efforts, orfollowing repeated rounds of manual selection, until the morphology ofthe tissue is suitable for regeneration, at least two weeks, thentransferred to media conducive to maturation of embryoids. Cultures aretransferred every two weeks on this medium. Shoot development willsignal the time to transfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, will then beallowed to mature into plants. Developing plantlets are transferred tosoilless plant growth mix, and hardened, e.g., in an environmentallycontrolled chamber at about 85% relative humidity, about 600 ppm CO₂,and at about 25-250 microeinsteins m⁻²·s⁻¹ of light. Plants arepreferably matured either in a growth chamber or greenhouse. Plants areregenerated from about 6 weeks to 10 months after a transformant isidentified, depending on the initial tissue. Dun ng regeneration, cellsare grown on solid media in tissue culture vessels. Illustrativeembodiments of such vessels are petri dishes and Plant Con®.Regenerating plants are preferably grown at about 19° to 28° C. Afterthe regenerating plants have reached the stage of shoot and rootdevelopment, they may be transferred to a greenhouse for further growthand testing.

Mature plants are then obtained from cell lines that are known toexpress the trait. If possible, the regenerated plants are selfpollinated. In addition, pollen obtained from the regenerated plants iscrossed to seed grown plants of agronomically important inbred lines. Insome cases, pollen from plants of these inbred lines is used topollinate regenerated plants. The trait is genetically characterized byevaluating the segregation of the trait in first and later generationprogeny. The heritability and expression in plants of traits selected intissue culture are of particular importance if the traits are to becommercially useful.

Regenerated plants can be repeatedly crossed to inbred maize plants inorder to introgress the preselected DNA sequence into the genome of theinbred maize plants. This process is referred to as backcrossconversion. When a sufficient number of crosses to the recurrent inbredparent have been completed in order to produce a product of thebackcross conversion process that is substantially isogenic with therecurrent inbred parent except for the presence of the introducedpreselected DNA sequence, the plant is self-pollinated at least once inorder to produce a homozygous backcross converted inbred containing thepreselected DNA sequence. Progeny of these plants are true breeding andthe weight percentage of a particular amino acid in a plant part, e.g.,the seeds, or the amount of starch in these progeny are compared to theweight percentage of that amino acid or amount of starch in therecurrent parent inbred, in the field under a range of environmentalconditions (see below). The determination of the weight percentage of anamino acid or amount of starch are well known in the art.

Alternatively, seed from transformed monocot plants regenerated fromtransformed tissue cultures is grown in the field and self-pollinated togenerate true breeding plants.

Seed from the fertile transgenic plants is then evaluated for thepresence and/or expression of the sense or antisense DNA sequence.Transgenic seed tissue can be analyzed for a substantial inhibition inthe production of the seed storage protein using standard methods suchas SDS polyacrylamide gel electrophoresis. A substantial inhibition ofthe production of the seed storage protein is a decrease in the weightpercent of the seed storage protein, preferably of about 70-100% andmore preferably about 80-100% over that normally present in anontransformed seed. The weight percent of a seed storage protein or anamino acid is based upon the amount of that protein or amino acidpresent per total weight of all proteins or amino acids in the seed. Theseed can also be evaluated for an increase in the weight percent of atleast one amino acid essential in the diet of animals by standardmethods. An increase in the weight percent of the target amino acid ispreferably about 50-300%, and more preferably about 100-200%, over thatnormally present in the untransformed seed. While not in any way meantto limit the invention, the decrease in the expression in the targetseed storage protein is generally accompanied by an increase in otherproteins having amino acids essential in the diet of animals.

Once a transgenic seed expressing the sense or antisense DNA sequenceand having an increase in the weight percent of the amino acid essentialin the diet of animals is identified, the seed can be used to developtrue breeding plants. The true breeding plants are used to develop aline of plants with an increase in the weight percent of an amino acidessential in the diet of animals as a dominant trait while stillmaintaining other desirable functional agronomic traits. Adding thetrait of increasing the weight percent of an amino acid essential in thediet of animals to agronomically elite lines can be accomplished byback-crossing with this trait and with those without the trait andstudying the pattern of inheritance in segregating generations. Thoseplants expressing the target trait in a dominant fashion are preferablyselected. Back-crossing is carried out by crossing the original fertiletransgenic plants with a plant from an inbred line exhibiting desirablefunctional agronomic characteristics while not expressing the trait ofan increased weight percent of the target amino acid. The resultingprogeny are then crossed back to the parent not expressing the trait.The progeny from this cross will also segregate so that some of theprogeny carry the trait and some do not. This back-crossing is repeateduntil the inbred line with the desirable functional agronomic traits,but without the trait of an increase in the weight percent of an aminoacid essential in the diet of animals, which is expressed in a dominantfashion.

Subsequent to back-crossing, the new transgenic plants are evaluated foran increase in the weight percent of an amino acid essential in the dietof animals as well as for a battery of functional agronomiccharacteristics. These other functional agronomic characteristicsinclude kernel hardness, yield, resistance to disease and insect pests,drought resistance, and herbicide resistance.

Plants that may be improved by these methods include but are not limitedto processed plants (canola, potatoes, tomatoes, lupins, sunflower andcottonseed), forage plants (alfalfa, clover and fescue), and the grains(maize, wheat, barley, oats, rice, sorghum, millet and rye). The plantsor plant parts may be used directly as feed or food or the amino acid(s)may be extracted for use as a feed or food additive.

C. Determination of Stably Transformed Plant Tissues

To confirm the presence of the preselected DNA sequence in theregenerating plants, or seeds or progeny derived from the regeneratedplant, a variety of assays may be performed. Such assays include, forexample, “molecular biological” assays well known to those of skill inthe art, such as Southern and Northern blotting and PCR; “biochemical”assays, such as detecting the presence of a protein product, e.g., byimmunological means (ELISAs and Western blots) or by enzymatic function;plant part assays, such as leaf, seed or root assays; and also, byanalyzing the phenotype of the whole regenerated plant.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR techniques may also be used for detection andquantitation of RNA produced from introduced preselected DNA segments.In this application of PCR it is first necessary to reverse transcribeRNA into DNA, using enzymes such as reverse transcriptase, and thenthrough the use of conventional PCR techniques amplify the DNA. In mostinstances PCR techniques, while useful, will not demonstrate integrityof the RNA product. Further information about the nature of the RNAproduct may be obtained by Northern blotting. This technique willdemonstrate the presence of an RNA species and give information aboutthe integrity of that RNA. The presence or absence of an RNA species canalso be determined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and will onlydemonstrate the presence or absence of an RNA species.

While Southern blotting and PCR may be used to detect the preselectedDNA segment in question, they do not provide information as to whetherthe preselected DNA segment is being expressed. Expression may beevaluated by specifically identifying the protein products of theintroduced preselected DNA sequences or evaluating the phenotypicchanges brought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focussing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as Western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of preselectedDNA segments encoding storage proteins which change amino acidcomposition and may be detected by amino acid analysis.

IV. Increasing the Weight Percent of at Least One Amino Acid Essentialto the Diet of Animals.

The present invention is directed to increasing the amount of an aminoacid essential to the diet of animals in a transgenic plant or seed overthat normally present in the corresponding nontransformed(nontransgenic) plant or its seed. Plant cells are stably transformedwith a preselected DNA sequence that encodes a RNA molecule havingsubstantial identity (sense), or complementarity (antisense), to a mRNAcoding for a seed storage protein, preferably a seed storage proteinwhich is deficient in at least one amino acid essential in the diet ofanimals. The transformed cells are used to regenerate fertile transgenicplants and seeds. The antisense, or sense, RNA sequence is expressed inthe seeds in an amount effective to inhibit the production of the seedstorage protein. The decrease in the seed storage protein deficient inthe essential amino acid results in an increase in the weight percent ofother amino acids, preferably essential amino acids, present in otherproteins in the transgenic seed over that normally present in thenontransformed seed.

In a preferred embodiment, a maize cell line is transformed with anexpression vector comprising a preselected DNA sequence coding for a RNAmolecule substantially identical, or complementary, to all or a portionof a mRNA coding for a 19 kD or 22 kD α-zein protein operably linked toa promoter for a 10 kD zein protein. Another preferred embodimentincludes linking the preselected DNA sequence to the Z27 promoter. Theexpression vector preferably further comprises at least one selectablemarker gene. The maize cell line is transformed by biolistictransformation and transformants are initially selected by growth in thepresence of an agent which is present at levels which inhibit the growthof the corresponding nontransformed cells. Transformants are furthercharacterized for the presence or expression of the preselected DNAsequence by polymerase chain reaction (PCR) or reverse transcriptase(RT-PCR) analysis. Transformed maize cell lines having the preselectedDNA sequence are used to regenerate fertile transgenic plants by themethod as described in PCT publication WO 95/06128. The fertiletransgenic plants are self-pollinated or crossed to a second plantvariety, and the transgenic seeds are characterized for the inhibitionof production of a 19 kD or 22 kD α-zein protein by quantitative Westernblot, or SDS-PAGE, and for an increase in the weight percent of an aminoacid essential to the diet of animals, such as lysine.

In an alternative embodiment, the present invention is directed toincreasing the weight percent of an amino acid essential in the diet ofanimals in a plant or seed by stably transforming the cells of a planttissue source with at least two different preselected DNA sequences. Thefirst preselected DNA sequence comprises a preselected DNA sequencecoding a RNA molecule substantially identical, or complementary, to amRNA for a seed storage protein, preferably a seed storage protein whichis deficient in at least one amino acid essential to the diet ofanimals. The second preselected DNA sequence encodes a polypeptidecomprising at least one amino acid essential to the diet of animals. Theexpression cassettes comprising one or both of the preselected DNAsequences can optionally comprise a selectable marker gene and,optionally, a reporter gene. Each preselected DNA sequence may comprisea different selectable marker gene so that transform ants containingboth preselected DNA sequences can be readily selected.

The cells of plant tissue source, as well as the methods oftransformation described previously, can be employed inco-transformation. Co-transformation can be conducted sequentially, thatis, the cells of plant tissue source can be transformed with the firstpreselected DNA sequence and transformants selected. The transformantscan then be transformed with the second preselected DNA sequence andtransformants having both preselected DNA sequences can be selected.Typically, the initial selection is based upon the trait expressed bythe selectable marker gene or genes. Co-transformation can also beconducted in one step, that is, the cells of the plant tissue source canbe transformed with both preselected DNA sequences at once, e.g., byelectroporation or biolistic transformation. Alternatively, two plantscan be crossed. The genome of one of the plants comprises the firstpreselected DNA sequence and the genome of the other plant in the crosscomprises the second preselected DNA sequence.

Transformants containing both preselected DNA sequences are furthercharacterized for the presence and/or expression of the firstpreselected DNA sequence and the second preselected DNA sequence bystandard methods, such as PCR or RT-PCR, Southern blot hybridization,SDS-PAGE and quantitative Western blot. Transformants having bothintroduced sequences are used to generate fertile transgenic plants andseeds therefrom as described previously.

The transgenic seeds are then characterized for the presence and/orexpression of both preselected DNA sequences. Expression of the firstpreselected DNA sequence can be detected and quantitated by examiningthe seeds for a substantial inhibition of the production of a seedstorage protein deficient in an amino acid essential in the diet ofanimals. Expression of the second preselected DNA sequence can bedetected and quantitated by quantitative Western blot for the plantprotein comprising at least one amino acid essential in the diet ofanimals and/or by an increase in the weight percent of an amino acidessential in the diet of animals, such as lysine or methionine, ascompared to an untransformed seed.

In a preferred embodiment, a maize cell line is co-transformed with afirst preselected DNA sequence coding for a RNA molecule substantiallyidentical, or complementary, to all or a portion of a mRNA coding for a19 kD or 22 kD α-zein protein, and a second preselected DNA sequencecoding for a 10 kD zein protein. The 19 kD or 22 kD α-zein protein ispreferably deficient in at least one amino acid essential in the diet ofanimals, such as lysine, methionine or tryptophan. The 10 kD zeinprotein preferably comprises at least one amino acid essential in thediet of animals, such as methionine. The isolated, purified DNA moleculecomprising the first preselected DNA sequence also preferably comprisesa selectable marker gene or a reporter gene, such as GUS. The secondpreselected DNA sequence may contain a second selectable marker gene,such as glyphosate resistant EPSPS.

In a further embodiment of the present invention, maize is cotransformedwith a first preselected sense DNA sequence coding for a RNA moleculewhich is identical, or complementary, to the 19 kD or 22 kD α-zein mRNAand a second preselected DNA sequence encoding the synthetic proteinMB1. Alternatively, the second preselected DNA sequence encodes a 27 kDzein protein. Thus, it is contemplated that genes encoding othersynthetic or naturally occurring proteins comprising at least one aminoacid essential in the diet of animals may be substituted for MB1. Evenmore preferably, maize is cotransformed with a first preselected senseDNA sequence coding for a RNA molecule which is identical, orcomplementary, to the 19 kD or 22 kD α-zein mRNA, a second preselectedDNA sequence encoding the synthetic protein MB1, and a third preselectedDNA sequence encoding a 27 kD zein protein.

Transformants having both preselected DNA sequences are used to generatefertile transgenic plants and seeds. The transgenic seeds arecharacterized by a substantial inhibition of the production of a 19 kDor 22 kD α-zein protein, determined, for example, by quantitativeWestern blot, and by an increase in the weight percent of an amino acidessential in the diet of animals, such as methionine or lysine. Thetransgenic seeds and plants can be used to develop true breeding plantsso that the trait of an increase of the weight percent of an amino acidessential in the diet of animals can be expressed as a dominant traitwhile still maintaining functional agronomic qualities, as describedhereinabove.

V. Method to Increase Starch Content of a Plant Seed

The invention also provides for an increase in the weight percent ofstarch in a plant and/or seed. The method comprises stably transformingthe cells of a plant tissue with a first preselected DNA sequence codingfor an RNA molecule substantially homologous or complementary to all ora portion of a mRNA coding for at least one seed storage protein. Whilenot in any way meant to limit the invention, it is believed that adecrease in the expression of seed storage protein in the seed resultsin an increase in the weight percent of the starch in the seed. Thepreselected DNA sequence is preferably operably linked to a promoterfunctional in a plant and/or seed. Transformed cells are used toregenerate fertile transgenic plants and/or seeds. The transgenic seedsare characterized for expression of the preselected DNA sequence byexamining the seed for a substantial inhibition of the production of atleast one seed storage protein and for an increase in the weight percentof starch over that normally present in an untransformed seed.

The first preselected DNA sequence can be derived from a DNA sequenceencoding at least one plant seed storage protein. Plant seed storageproteins include the zein proteins of maize such as the α-, β-, γ-, orδ-zein proteins. While not in any way meant to limit the invention, itis believed that a decrease in the expression of seed storage protein inthe seed results in an increase in the weight percent of the starch inthe seed. Preferably, the presence of the first preselected DNA sequenceresults in a substantial inhibition of at least one seed storageprotein, and more preferably results in the inhibition of the α-zeinproteins. The preparation of said first DNA sequence as well as itslinkage to suitable promoters can be accomplished as describedhereinabove. Cells of plant tissue can be transformed as describedabove, and transformants selected. Transformants are used to generatefertile transgenic plants and seeds.

Transgenic seeds are characterized by an increase in the weight percentof starch in the seed over that present in the untransformed seed. Theweight percent of the starch content in the seed can be determined byenzymatic hydrolysis and glucose determination. The weight percent ofstarch is calculated by comparing the weight of the starch in the seedcompared to the total weight of the seed. An increase in the weightpercent of the starch in the transgenic seed is preferably about 1 to10%, and more preferably about 3 to 8%, and even more preferably about 5to 7%, over that in the non-transformed seed.

Transgenic seeds with an increase in the weight percent of starch can beused to develop true breeding plants expressing this trait in a dominantfashion while still maintaining functional agronomic traits as describedpreviously.

Reduction of α-zein levels in corn kernels may also increase the degreeof starch recovery from operations such as wet-milling of grain asα-zeins constitute the major portion of the proteinaceous matrix whichsurrounds starch granules in the kernel (Lopes and Larkins, 1993). Areduction in the amount of these hydrophobic proteins could facilitaterecovery of starch grains. This is of particular significance forspecialty starches, such as that obtained from high-amylose corn or waxycorn, because those starches are of much higher value than that obtainedfrom No. 2 yellow dent corn. An increase in starch yield, i.e., thepercent of starch present in the kernel which may be recovered by wetmilling, is preferably about 1% to 20%, more preferably about 3% to 15%,and even more preferably about 6% to 12%, greater in grain from plantscontaining the preselected DNA sequence over grain from plants which donot contain the preselected DNA sequence.

VI. A Method for Inhibiting the Expression of a Family or Subfamily ofSeed Storage Proteins.

The invention also provides a method for inhibiting the expression of afamily, or subfamily, of seed storage proteins. Seed storage proteinssuch as the maize zein proteins are encoded by multi-gene families. Themulti-gene families corresponding to zein proteins have differentmolecular weights: α-zein proteins include proteins with molecularweights of 19 kD and 22 kD; β-zein proteins include proteins with amolecular weight of 14 kD; γ-zein proteins include proteins withmolecular weights of about 27 kD and 16 kD; and δ-zein proteins includeproteins with molecular weights of about 10 kD. Each family can haveseveral subfamilies. For example, the subfamilies for α-zein proteinsare determined on the basis of sequence homology to cDNA clones A20,A30, B49, B59, or B36 as described by Messing et al., cited supra., orthe Z4 cDNA clone encoding the 22 kD α-zein. Typically, members of thesame subfamily share about 90% to 100% amino acid sequence homology andmembers of different subfamilies share about 60% to 80% amino acidsequence homology.

The examination of the amino acid sequence for the α-zein subfamilieshas identified four functional subdomains and regions of shared aminoacid homology in these functional subdomains as shown in FIG. 1. Theseregions of amino acid sequence homology can be used to analyze aminoacid sequences from other subfamilies and families of zein proteins forhomology. In addition, these regions can be used to select DNA sequencesthat encode a RNA molecule that can inhibit production of a family or asubfamily of the zein proteins. An antisense RNA sequence than caninhibit production of a family or subfamily of zein proteins ispreferably a sequence that is substantially complementary to a portionof a mRNA sequence that is substantially homologous between all membersof the subfamily or family of the zein proteins. Alternatively, it iscontemplated that preselected sense DNA sequences may be used tosuppress the synthesis of a family or a subfamily of zein.

For example, as shown in FIG. 1, the A20, A30, and B49 subfamilies shareamino acid sequence homology in the signal peptide region and aminoterminal region of the proteins. An antisense DNA sequence encodingthese regions of the zein protein can encode a RNA molecule that caninhibit expression for a family of zein proteins. The antisense DNAsequence encoding these regions can be selected based on the amino acidsequence homology in these regions and can be used to inhibit expressionof more than one subfamily of a family of the zein proteins.

The domain containing the tandem repeats of 20 amino acids has thegreatest variability in amino acid sequence and size. There areinsertions and deletions in this region when the sequence of differentsubfamilies are compared. A preselected antisense DNA sequence encodingthis region of the α-zein protein can be employed to express a RNAmolecule that can inhibit the expression of a subfamily of zeinproteins. The preselected antisense DNA sequence from this region of thezein protein is substantially homologous within a subfamily but is notsubstantially homologous between subfamilies.

The preselected antisense DNA sequence is obtained by restrictionendonuclease digestion of a cDNA or genomic clone coding for a seedstorage protein. The preselected antisense DNA sequence is linked to apromoter to form an antisense expression cassette to determine thecapacity of the antisense DNA sequence to inhibit translation of afamily or subfamily of seed storage proteins. A standard assay such ashybrid arrested translation may be employed. The preselected antisenseDNA sequence results in substantial inhibition of translation of cDNAclones from several families such as A20, Z4, A30, and/or B49. Thepreselected antisense DNA sequence can inhibit a family of zeinproteins. The preselected antisense DNA sequence substantially inhibitstranslation of cDNA clones or genomic clones within a subfamily and thepreselected antisense DNA sequence can be used to inhibit expression ofa subfamily of zein proteins. The preselected antisense DNA sequence isused to stably transform plant cells as described hereinabove. Sense DNAsequences may also be used. Fertile transgenic plants and seeds aregenerated from the transformed cells.

The transgenic seeds are characterized for expression of the preselectedantisense DNA sequence by evaluating inhibition of production of two ormore members of a family or subfamily of zein proteins by usingtechniques such as quantitative Western blot.

In a preferred embodiment, the preferred antisense DNA sequence codingfor a RNA molecule substantially complementary to a mRNA coding for thetandem repeat region of domain 3 of an α-zein protein in A20 subfamilyis combined with a Z10 promoter. The expression cassette comprising thepreselected antisense DNA sequence can also comprise one or moreselectable marker genes. The preselected antisense DNA sequence isstably transformed into a maize cell line and transformants areselected. Transformed cells are used to generate fertile transgenicplants and seeds. The transgenic seeds are evaluated for expression ofthe preselected antisense DNA sequence by conforming substantialinhibition in the production of the A20 subfamily of α-zein proteins byquantitative Western blot.

VII. Method for Increasing the Production of a Preselected Polypeptidein Seeds

The invention further provides for an increase in the expression of aparticular polypeptide in plants and/or seeds. The method involvesstably transforming cells with a first preselected DNA sequence tosuppress synthesis of a seed storage protein deficient in an essentialamino acid and a second preselected DNA sequence coding for apolypeptide, such as an enzyme or a seed storage protein. While not inany way meant to limit the invention, it is believed that a substantialinhibition of production of at least one seed storage protein isaccompanied by an increase in the capacity of the plant cell and/or seedto produce other proteins. Transformed cells having both first andsecond preselected DNA sequences are obtained and used to generatefertile transgenic plants and/or seeds.

The first preselected DNA sequence encodes an antisense or sense RNA forat least one seed storage protein. The first preselected DNA sequence iscombined with a promoter functional in plant and/or seed to form anexpression cassette. Optionally and preferably, the expression cassettealso comprises a selectable marker gene and, optionally, a reportergene.

The second preselected DNA sequence, which encodes a polypeptide, isoperably linked to a promoter functional in plant and/or seed.Preferably, the promoter is functional during plant and seeddevelopment. The second preselected DNA sequence encodes a polypeptidethat provides the plant or seed with a desirable functionalcharacteristic, such as increased disease or pest resistance, droughtresistance, increased amino acid biosynthesis, increased nutritionalvalue, increased kernel hardness, and the like.

The preselected DNA sequences can be operably linked to the promoter bystandard methods provided in Sambrook et al., cited supra., and asdescribed previously. Optionally and preferably, the expression cassettewhich comprises the second preselected DNA sequence also comprises aselectable marker gene different from the selectable marker gene presentin the expression cassette comprising the first preselected DNAsequence.

Transformation of plant cells is conducted by any one of the methodsdescribed previously. The plant cells can be transformed with the firstand/or second preselected DNA sequences sequentially or simultaneously.When the plant cells are sequentially transformed, transformantscomprising the first preselected sequence are obtained based upon thepresence of a selectable marker gene. These transformed cells are thentransformed with the second preselected DNA sequence and transformantsare obtained based upon the presence of each of the selectable markergenes present on the expression cassette comprising the firstpreselected DNA sequence and present on the expression cassettecomprising the second preselected DNA sequence. Transformants containingboth the first and second preselected DNA sequences are used toregenerate fertile transgenic plants and/or seeds.

The transgenic seeds are characterized by expression of the first andsecond preselected DNA sequences. Expression of the first preselectedDNA sequence is evaluated by measuring a substantial inhibition in theproduction of at least one seed storage protein. Expression of thesecond preselected DNA sequence is evaluated by detecting thepreselected polypeptide using standard phenotypic or genotypic methods,such as quantitative Western blot. An increase in the expression of apolypeptide can be determined by comparing the weight percent of theprotein produced in plants or seeds transformed with the secondpreselected DNA sequence. The expression of the polypeptide ispreferably increased about 2- to 100-fold, and more preferably about 5-to 30-fold, over that in a plant and/or seed only transformed with thesecond preselected DNA sequence.

The invention will be further described by the following examples.

EXAMPLE 1 Construction of Plasmid Containing Antisense DNA Constructs

Antisense expression cassettes were obtained by using sequences fromcDNA clones encoding zein proteins. The cDNA clones were prepared bystandard methods, described previously by Geraghty et al. (1982) and Huet al. (1982), which are hereby incorporated by reference. The cDNAclone A20 encodes an α-zein protein of the 19 kD size class of the Z1Asubfamily of zein genes. Another cDNA clone designated Z4 encodes anα-zein of the 22 kD size class of the Z1B family of genes. The Z1A andZ1B subfamilies and their characteristics are shown in Table I.

TABLE I Prolamin Fraction of Maize (Alcohol Soluble) Zein MultigeneFamily z1 z2 (non-reducing conditions) (reducing conditions) Subfamilyz1A z1B z1C z1D z2A (ASC) z2B z2C Representing cDNA A20 A30 B49 B59 B36Z15A — clone Mr × 100 mostly 19 mostly 19 mostly 22 mostly 19 27 15 10some 22 some 19 Locus 4L, 7S, 10L 4L, 7S 4L ? ? ? ? Predominant aminoglutamine glutamine glutamine glutamine proline cysteine methionine acidTiming of expression ca 12 dap ca 12 dap ca 18 dap ca 12 dap ca 18 dapca 18 dap ca 18 dap Transacting mutants o2+.o7+++ o2+.o7++ o2+++.o7++o2+.o7++ o2+.o7+ o2+.o7++ o2++.o7++ (o6+++.f12+.Mc+) De*-B30+ De*-B30+De*-B30+++ De*-B30+ De*-B30+ De*-B30+ De*-B30− No. of genes <25 <20 <15<5 2 2 ? + reduced synthesis ++ increasingly reduced synthesis +++strongly reduced synthesis

Antisense expression cassettes comprising the complete cDNA sequence forclones A20 and Z4, as well as portions of those sequences, weregenerated. The portions of each sequence were selected by examining thcsequence of the 19 kD and 22 kD α-zein proteins. As shown in FIG. 1, theprimary sequence of the polypeptides can be divided into four domains,as described by Messing et al. (1983). Domain I contains the highlyconserved 21 amino acid signal peptide that is cleaved duringcotranslational transport of zein proteins into the lumen of theendoplasmic reticulum. Domains II and IV are the N-terminal andC-terminal regions, respectively, of the mature zein proteins. DomainIII represents the major source of sequence homology between subfamiliesas it contains 9-10 tandem repeats of sequence encoding a 20 amino acidsequence. The number of repeats present in Domain III determines thesize of the α-zein protein (19 kD or 22 kD). Typically, individualmembers within a subfamily share 90-100% sequence homology and while thesequence homology between subfamilies ranges from about 65-85%.

All antisense plasmids for in vitro system analysis were constructed bystandard recombinant techniques as detailed below, using thetranscriptional vectors pSP72 and pSP73 (Promega, Madison, Wis.). Thesetranscription vectors are 2.46 kb circular plasmids, containing 103 bpof polylinker sequence inserted between convergent T7 and SP6transcriptional promoters. The two transcription vectors differ in theorientation of the polylinker with respect to the promoters. Antisenseplasmids, complementary to all or portions of the cDNA clones A20 andZ4, were constructed as described below.

The RNA sequence for A20 (SEQ ID NO:1) and the DNA sequence for Z4 (SEQID NO:2) zein are shown in FIGS. 2 and 3, respectively. The relevant A20and Z4 genes and gene fragments used in antisense constructs are shownin Table II.

TABLE II Antisense Construct Restriction Insert Designation Enzymes SizeSP20 ent BalI/EcoRI 711 SP20R3′ BalI/PstI 488 SP20R PstI/PstI 262 SP20PBalI/EcoRI 863 SP20P5′ AccI/EcoRI 458 SPZ4ent SacI/BamHI 960 SPZ4R3′XbaI/BamHI 713 SPZ4R5′ BamHI/DdeI 246 SPZ10ent EcoRI 640

All restriction and modification enzymes and buffers were obtained fromNew England Biolabs, Inc. (Beverly, Mass.), unless otherwise noted, andused according to the manufacturer's specifications. All insertfragments were gel isolated and purified by the Geneclean method (BIO101, Vista, Calif.), and all vectors were treated with calf intestinalphosphatase (Boehringer-Mannheim Corporation, Indianapolis, Ind.), thengel isolated on low melting point agarose before addition to theligation reactions.

Antisense constructs encoding all or a portion of the cDNA clones fromA20 and Z4 were prepared as follows:

SP20ENT: The parent plasmid pUC12/A20, containing the entire maturecoding and 3′ nontranslated sequence (nts) from the A20 cDNA clone (theRNA sequence of A20 is shown in FIG. 2), was digested at the EcoRI site(nt 175) and the BalI site (nt 886) to generate a 711 nt fragmentcontaining the entire sequence except for 55 bp of 3′ nts. The fragmentwas ligated into pSP72 which had been digested with EcoRI and PvuII,resulting in 3′ to 5′ antisense orientation of the gene with respect tothe SP6 promoter.SP20R3′: A 488 bp fragment, containing the sequence encoding themid-repeat region through the 3′ nts A20 from the Pst I site at nt 298to the BalI site at nt 886, was isolated from the parent plasmidp1020R3′ prepared as in Example 2. The fragment was obtained bydigesting p1020R3′ with KpnI and HindIII, and after isolation thefragment was ligated into pSP72 that had been digested with theseenzymes also. The gene fragment was therefore oriented 5′ to 3′ withrespect to the SP6 promoter.SP20R: A 262 bp fragment, from nt 398 to nt 660 was obtained bydigesting pUC12/A20 with PstI. The purified fragment was ligated intopSP72 digested with PstI to make pSP20R, containing the sequenceencoding the mid-repeat region of A20 in the 3′ to 5′ orientation withrespect to the SP6 promoter.SP20P: The 5′ end of the A20 transcription unit was reconstructed by PCRamplification of a fragment containing 5′ nts and encoding the signalpeptide through the mid-repeat region, since the 5′ nts and signalpeptide sequence was not contained in the pUC12/A20 clone. The primersused in the amplification are designated A20P5′.2 (SEQ ID NO:3) andA20P3′ (SEQ ID NO:4). The fragment was amplified from genomic DNAisolated from leaf tissue from the maize inbred line A654, and contained458 bp of A20 cDNA sequence, from nt 58 to nt 490.

The conditions for PCR are detailed below; all reactions were carriedout in a Biosycler™ oven (Bios Corporation). Each reaction contained 10μl of 10×PCR reaction buffer, 10 μl of 20 mM MgCl₂, 10 μl of 2 mM dNTPs,10 μl of each primer (stock 2.5 ml) and 0.5 μl (2.5 U) of Taq polymerase(Perkin-Elmer Cetus), for a total of 100.5 μl/reaction. An annealingtemperature of 56° C. was used, and a total of 30 cycles were performed,including the first three cycles with extended incubation at the 94° C.denaturing temperature. Parameters for the first three amplificationcycles were as follows: 60 seconds at 94° C., 30 seconds at theannealing temperature of 56° C., and 30 seconds at the synthesistemperature of 72° C. For the remaining 27 cycles, the parameters wereas follows: after bringing the reactions to 94° C., 15 seconds at thistemperature, then 15 seconds at 56° C., followed by 15 seconds at 72° C.

The 458 bp product was designed to add a 5′ EcoRI site, and included anendogenous 3′ AccI site. After digestion with these enzymes, theamplified fragment was ligated into pSP20ENT also digested with theseenzymes, replacing a 320 bp fragment containing the shorter 5′ endfragment of A20 from pSP20ENT. After reconstruction, the gene wasapproximately 860 bp long, and contained approximately 55 nt of 5′ nts,the sequence encoding the signal peptide, and the entire coding sequenceas well as 3′ nts. The reconstructed gene is oriented 3′ to 5′ withrespect to the SP6 promoter.

SP20P5′: The 5′ end of the A20 gene, after PCR amplification anddigestion with EcoRI and AccI as described above, was cloned into pSP72to generate pSP20P5′. This construct contains 458 nt of A20 sequence,including 55 nt of 5′ nontranslated sequence and 403 nt of codingsequence, which includes approximately the N terminal half of the codingsequence. The inserted sequence is oriented from 3′ to 5′ with respectto the SP6 promoter.SPZ4ENT: Essentially, the entire Z4 transcription unit is contained inthis clone, with a total insert size of 960 nt. The gene wasreconstructed from two Z4 subclones, pSPZ4R3′ and pSPZ45′, which aredescribed below. The parent vector was pSPZ4R3′, containing 713 nt ofmid-repeat to 3′ nts sequence, from nt 630 to nt 1341 of the Z4 sequence(the DNA sequence of Z4 is shown in FIG. 3). The 5′ end of the Z4sequence was released by digestion with SacI (which cleaves thepolylinker sequence outside the inserted gene) and BamHI, and the insertcontaining the 5′ sequence from pSPZ45′, obtained by SacI (which alsocleaves the polylinker sequence) an dBamHI digestion, was ligated to thelinearized pSPZ4R3′, resulting in reconstitution of the intact Z4transcription unit.SPZ4R3′: A 713 nt insert fragment, containing the mid-repeat region tothe 3′ noncoding sequence, was isolated after digestion with BamHI (nt630) and XbaI (nt 1341). The fragment was ligated into pSP72 digestedwith the same enzymes, resulting in orientation of the gene fragment in3′ to 5′ direction with respect to the SP6 promoter.SPZ45′: A 247 nt fragment containing 76 nt of 5′ noncoding sequence, thesignal peptide sequence, and approximately 100 nt of mature proteincoding sequence was cloned into pSP72. After digestion with DdeI, theDNA was Klenow treated to create blunt ends, then digested with BamHI torelease the desired fragment. The fragment was ligated into pSP72digested with EcoRV and BamHI, resulting in 3′ to 5′ orientation of thegene fragment with respect to the SP6 promoter.SPZ10ENT: A 670 nt fragment containing the entire Z10 transcriptionalunit was isolated from the 10 kD zein cDNA clone p10kZ-1 by digestionwith EcoRI (the sequence of the 10 kD zein gene can be found in Kiriharaet al., 1988). After digestion of pSP72 with EcoRI also, the insert wasligated with the vector to produce pSPZ10ENT, a circular plasmid of 3.16kb. Clones were obtained containing both orientations, and the cloneused in the hybrid arrest studies contained the 10 kD transcription unitoriented 3′ to 5′ with respect to the SP6 promoter.

EXAMPLE 2 Construction of Plasmids Containing an Antisense DNA Sequencefor use in Maize Transformation

A set of antisense plasmids was constructed for expression in maize,using entire or portions of the Z4 and A20 sequence as detailed inExample 1, above. The antisense constructs were combined with a promoterfunctional in plant endosperm tissue to form a DNA sequence that can beexpressed in a plant seed.

Vector Construction

The plasmids p10B and p10X were constructed from pZ10nos3′. Theconstruct pZ10nos3′ contains 1137 bp of the Z10 promoter from a geneencoding a 10 kD zein promoter upstream of a short polylinker, which isadjacent to the nos poly A 3′ element. The vectors p10X and p10B werecreated by digestion of pZ10nos3′ with BamHI, Klenow treatment to bluntthe BamHI site, then ligation with a polylinker insert, resulting inclones containing both orientations of the polylinker with respect tothe Z10 promoter. The polylinker fragment was obtained by digestingpSP73 with BglIII and XhoI, followed by Klenow treatment then ligationwith the prepared pZ10nos3′ vector. The p10X version contains thepolylinker oriented with the XhoI site proximal to the Z10 promoter,while the p10B version contains the polylinker oriented with the BglIIIsite proximal to the Z10 promoter. Both plasmids are circular plasmidsof approximately 4.65 kb. Antisense DNA expression constructs, preparedas described in Example 1 were combined with a promoter functional in aplant seed utilizing the p10B and p10X plasmids, as described below.

1020ENT: A 725 nt insert fragment containing the mature A20 coding and3′noncoding sequence (see Example 1, SP20ENT section), and includingsome polylinker sequence, was obtained by digestion of SP20ENT with ClaI(cuts in the polylinker sequence) and XhoI. The vector, p10X, wasprepared by digestion with ClaI and XhoI also, then the insert andvector were ligated, generating p1020ENT, which contains the A20sequence inserted 3′ to 5′ with respect to the Z10 promoter.1020R3′: A 488 nt insert fragment, containing the mid-repeat to the 3′noncoding sequence of A20, was isolated from the clone pUC 12/A20. Theinsert contains sequence from the PstI site at nt 398 and continues tothe Ball site at nt 886. The insert was obtained by digestion ofpUC12/A20 with Hind III which cuts outside the A20 sequence, thenpartial digestion with PstI (digestion only at the nt 398 PstI site),followed by gel isolation of the desired fragment of 740 nt. Afterpurification, the HindIII/PstI fragment was digested with Ball, whichremoved approximately 252 nt from the 3′ end to generate a 488 ntfragment with PstI/Ball ends. This fragment was ligated into p10B whichhad been cut with SmaI and PstI, resulting in insertion of the A20R3′fragment in the 3′ to 5′ orientation with respect to the Z10 promoter.1020R: A 262 nt insert fragment, containing the mid-repeat region fromA20 (as in SP20R from Example 1), was obtained by digestion of pUC12/A20with PstI. The vector p10X was also digested with PstI and, afterligation, clones were obtained with both orientations of the fragmentwith respect to the Z10 promoter. An asymmetrical AccI site within theinsert was used to select clones containing the fragment in the desiredantisense orientation.pDPG380: The 863 nt insert fragment containing the reconstructed A20gene (as described for pSP20P above) was obtained by digesting pSP20Pwith XhoI and BglII (both of which cut in the polylinker), then ligatingthe fragment into p10X that had been digested with XhoI and BamHI. Thisresulted in a 3′ to 5′ orientation of the reconstructed A20 gene withrespect to the Z10 promoter.pDPG340: A 875 nt fragment, containing the entire Z4 gene as describedabove for pSPZ4ENT, was obtained by digestion of pSPZ4ENT with HindIII,Klenow treatment, then digestion with SalI. These enzymes cut in thepolylinker sequence outside the gene in pSPZ4ENT. The vector, p10X, wasdigested with NcoI, Klenow treated, then digested with XhoI beforeligation with the insert fragment. The resulting clone contained thegene in 3′ to 5′ orientation with respect to the Z10 promoter.10Z4R3′: An insert of approximately 750 nt, consisting of the Z4mid-repeat through the 3′ noncoding (as described in Example 1 forpSPZ4R3′) was obtained by digesting pSPZ4R3′ with SacI and SalI, whichcut in the polylinker sequence. The vector, p10X, was digested with SalIand XhoI, and since XhoI and SalI create compatible ends, this resultedin directional cloning of the Z4R insert in the 3′ to 5′ orientationwith respect to the Z10 promoter.10Z45′: An intermediate vector 119Z45′, containing the Z45′ sequenceinsert (see SPZ45′ construction, Example 1) was first constructed usingthe pUC119 backbone (Sambrook et al., 1989).

The final construct, 10Z45RN, was constructed by moving the Z45′ insertfrom 119Z45′ into the p10B vector. First, 119Z45′ was digested withBamHI and PstI, releasing a 270 bp fragment. The vector, p10B, wasprepared by digestion with BamHI and PstI, and then the vector andinsert were ligated to produce p10Z45′, containing the Z45′ insert inthe antisense orientation with respect to the Z10 promoter.

pDPG530 and pDPG531: pDRG530 and pDRG531 were made by cutting a fragmentof approximately 960 bp from SPZ4Ent and filling in the ends. The vectorwas a Z27promoter::Nos 3′ region construct in pBSK(−) which contained aunique NcoI site between the promoter and terminator. Both the vectorand insert were blunt-ended and ligated. Clones were identified with thesense orientation of the Z4 DNA sequence (pDPG531) and the antisenseorientation of the Z4 DNA sequence (pDPG530).

EXAMPLE 3 In Vitro Method for Screening Antisense Containing DNASequences

Once an expression cassette comprising a preselected antisense DNAconstruct and a promoter functional in a plant seed was prepared, asdescribed in Example 2, the expression cassette was screened for theability to arrest translation of the genes encoding the 19 kD (A20) and22 kD (Z4) α-zein proteins. The expression cassettes comprising theantisense DNA sequences were screened by standard hybrid arrestedtranslation, as described below.

Template production. All reagents for in vitro transcription wereobtained from Promega (Madison, Wis.), using their SP6/T7 transcriptionprotocol. Slight modifications were made to the Promega protocol.Plasmids were digested with appropriate enzymes in order to linearizethe templates, preventing transcription beyond the end of the insertedgene. Templates were digested with XhoI for SP6 transcription, and withBglII for T7 transcription, unless otherwise noted.

Twenty micrograms of DNA were digested in a total volume of 100 μl.After analyzing aliquots for complete digestion, digests were extractedwith phenol/chloroform and chloroform, then precipitated with 0.1volumes 3M sodium acetate, 2.5 volumes ethanol. After washing with 70%ethanol, pellets were resuspended in 10 μl of sterile, RNase-free water.

Transcription reactions. After thawing all reagents at room temperature,master transcription mixes were prepared, excluding template DNA. Thisresulted in greater yield uniformity of the reactions. For eachreaction, the following components were added to 5 μl of template DNA at1 μg/μl in RNase-free water: 20 μl of 54 transcription buffer, 10 μl at0.1 M DTT, 2.5 μl of recombinant RNasin (an RNAse inhibitor supplied at40 U/μl), 20 μl of 10 mM rNTP mix, 2.5 μl of SP6 or T7 (20 U/μl), and 45μl of RNase-free water. Reactions were incubated at 37° C. for two hoursbefore template removal. Templates were removed by digestion with RQ1DNase (1 U/μl), 5.0 μl of enzyme was added to the transcriptionreactions, which were then incubated at 37° C. for 15 minutes beforeextraction and precipitation of the transcript. Extraction,precipitation and washes were performed as described above for templatepreparation.

Transcript yield was determined by absorbance readings at 260 nm, andintactness of the preparations was determined by gel analysis, eithernative or denaturing. Although native gels occasionally showed bands ofanomalous mobility, generally transcript preparations exhibited aroughly linear relationship between the expected transcript size andtheir mobility on native gels.

Annealing of Transcripts for Hybrid Formation. Before translation,transcripts were allowed to anneal under controlled temperature and saltconditions, using constant molar ratios of sense to antisensetranscript. Conditions for annealing were as follows: 10 mM Tris, pH7.5, 100 mM NaCl, RNA(s), and RNase-free water to bring the total volumeto 20 μl. The amount of RNA added was based on a 4:1 molar ratio ofantisense to sense transcript, with 4 μg of sense transcript in eachreaction, and a variable μg amount of antisense transcript added tomaintain the 4:1 molar ratio.

Before annealing, all transcripts were heated to 65° C., then kept at 0°C. to reduce potential formation of intramolecular secondary structureswhich would reduce the efficiency of duplex formation. After annealingfor 45 minutes at 45° C., the reaction was split in half, so that 10 μlof the reaction could be translated in vitro, and the remaining 10 μlwas analyzed on 1.2% agarose gels to determine the extent of hybridformation in each sample. Although some anomalies in mobility were seenthat were probably due to intramolecular interaction, this method wasgenerally useful for analyzing the extent of duplex formation betweentwo transcripts, and correlated well with the hybrid translationresults.

In Vitro Translation of Annealing Reactions and Analysis of TranslationProducts.

Translation of both Z4ENT and A20 ENT transcripts was performed usingwheat germ lysate and rabbit reticulocyte lysate systems (Promega).Although both systems produced detectable protein when the products oftranslation were analyzed by SDS-PAGE and autoradiography, the rabbitreticulocyte system translated both the Z4ENT and A20ENT transcriptsmore efficiently than the wheat germ system.

Translation of the annealed samples was performed in vitro, using anuclease-treated rabbit reticulocyte lysate system (Promega), and ³⁵Smethionine was used to label the translation products (Amersham). Thereactions were performed essentially according to the Promega protocolwith modifications as described below.

To analyze translation products, reactions were run on SDS-PAGE, using a4% stacking gel and 15% separation gel, with 0.75 or 1.5 mm spacers.Gels were run on a Hoefer apparatus, at 35 mA with constant current, for3 to 3.5 hours. Samples were prepared for electrophoresis by adding 10μl of each reaction to 40 μl of 1× sample buffer, then boiling for 7minutes before spinning for 30 seconds in a microfuge. After removal ofthe stacking gel, gels were incubated for 30 minutes with shaking in asolution of 1 M sodium salicylate to enhance detection of theradioisotope. Gels were then rinsed briefly in water and dried on a slabdrying under vacuum, at 65° C. for 2 hours. The dried gels were exposedto film overnight, using intensifying screens (Lightning Plus, DupontCronex) at −70° C. After developing, the gels were scanned using an LKB2202 Ultroscan laser densitometer, and the data was compiled andanalyzed using the Maxima software for chromatographic analysis (WatersCo.).

The results of in vitro translation of linearized plasmids containingthe complete copies of the Z4 and A20 genes in the sense orientationshow that the in vitro translation systems could be used to monitor theeffects of antisense constructs on translation of the zein genes. Bothtranslation systems produced proteins of the expected 19 kD weightspecies corresponding to the mature A20 gene product. Interestingly,however, while the rabbit reticulocyte system translated the Z4ENTtranscript into the 22 kD preprotein, the wheat lysate system processedthe Z4 preprotein, removing the signal peptide to produce the maturezein, resulting in a protein of approximately the same size as the 19kD. In both systems, translation of the A20ENT transcript was at least2-5× more efficient than translation of the Z4ENT transcript, probablydue to the lack of a signal peptide in the A20ENT protein or differencesin accessibility of the start codon between the two transcripts, sincethe A20ENT transcript did not contain 5′ noncoding sequence.

Capping of the Z4ENT and A20ENT transcripts was performed as a possiblemeans of increasing translation efficiency, using both cotranscriptionaland posttranscriptional procedures. No increase in translationefficiency was observed with either method.

Hybrid arrest translations were performed using Z4ENT sense transcriptsand Z4ENT antisense transcripts to establish annealing and translationconditions. A titration experiment was performed to determine the ratioof antisense:sense transcripts needed to completely abolish Z4synthesis. Amounts of antisense transcript were added to 1, 2, and5-fold excess over the amount of sense transcript and allowed to annealunder controlled conditions. Results of this experiment are shown inTable III. Subsequent experiments, using a 4:1 ratio of antisense:sensein the annealing reactions, were found to eliminate Z4 synthesis also,and so this ratio was used for later experiments.

TABLE III Effect of Increasing the Ratio of Antisense to SenseTranscript on Z4 Synthesis % Reduction in Z4 Synthesis Transcripts RatioRange Mean Z4ENTs na na na Z4ENTas/Z4ENTs 1:1 55-63 59 Z4ENTas/Z4ENTs2:1 84-85 85 Z4ENTas/Z4ENTs 5:1 100 100 

Experiments were also done to determine whether the radiation dose/filmexposure plot was sufficiently linear to allow quantitation of proteinusing laser densitometer readings of the film. To test this, the amountof extract loaded per lane was varied over a 25-fold range. Resultsindicated that the dose/response plot was acceptable over a 10-foldrange only. Densitometry of the autoradiograms indicated that anovernight exposure of gels to film produced a meaningful dose-responsecurve, but that longer exposures did not.

Having established a basic protocol using the complete, perfectlycomplementary Z4ENT sense and antisense transcripts, a series ofexperiments was initiated to compare these results with the effect ofantisense transcripts made from constructs containing only a portion ofthe Z4 transcriptional unit, as well as with antisense transcripts madefrom constructs containing all or portions of the A20 transcriptionalunit. Data was compiled from several hybrid arrest of translationexperiments, all performed using a 4:1 molar ratio of antisense sensetranscript, and all incorporating the Z4ENT sense transcript with noantisense transcript added as a negative control (representing 100%synthesis of Z4, or 0% reduction in Z4 synthesis), and the Z4ENTtranscript with the Z4ENT antisense transcript added as a positivecontrol (representing 100% reduction in Z4 synthesis). A lambdatranscript and a polylinker transcript were used as controls. Theresults are shown in Table IV.

The results are shown in Table IV.

TABLE IV Hybrid Arrested Translation Compiled Densitometer Data forReduction in Z4 Protein Synthesis Number of Antisense Mean ExperimentsTranscript Reduction (%) Performed Z4ENT 100  5 Z45′ 80 3 Z4R3′ 75 3A20ENT 81 3 A20R 59 2 Z10E 42 2 lambda transcript 32 1 polylinkertranscript  0 2

General conclusions about the results can be drawn by summing the entiredata set to generate a single rough consensus for efficiency of theantisense transcripts in effecting shutdown of Z4 synthesis, which areas follows:

-   -   Z4ENT>Z45′>A20ENT>Z4R3′>A20R>>Z10ENT>lambda>polylinker.        This data indicates that the entire complementary transcript, as        expected, is most efficient at reducing translation, and that        antisense transcripts annealing to the translation initiation        sequence are generally more efficient than transcripts annealing        to the downstream coding region.

EXAMPLE 4 Production of Reagent Antibodies for Analysis of MaizeTransformants

In order to screen for effects of antisense gene expression on zeinexpression levels in transformed cell lines and plants, polyclonalantibodies reactive with both the targeted α-zeins and with total zeinswere produced. Antigens were extracted and purified as described belowbefore inoculation into rabbits and subsequent antiserumcharacterization.

A. Antigen Purification

Total zeins were obtained by extraction of the maize inbred line BSSS53.In this procedure, 4 grams of dry kernels were ground to a fine powderin a Braun coffee mill, defatted by incubation with 15 ml/g of acetone,with stirring, for 90 minutes at room temperature. The defatted meal wasthen filtered through a Buchner funnel and allowed to dry. Twoextractions with 10 ml/g of 0.5 M NaCl were then performed; the mixturewas stirred at room temperature for 30 minutes before filtering asabove. Finally, two extractions were performed on the meal with 10 ml/geach of 70% ethanol % BME, for 60 minutes each, at room temperature withstirring. The ethanol extracts, totaling 80 ml, were pooled and filteredthrough a 0.45 micron filter before reducing the volume in a rotaryevaporator (Rotovapor R110, Buchi Corp.). Evaporation was performed at65° C., and after approximately 45 minutes the volume was reduced to 20ml of solution, which had a cloudy appearance. This solution was dilutedto 40 ml with sterile deionized water before freezing andlyophilization. A dry weight of 329 mg was obtained, and a 1 mg samplewas weighed out, resuspended in 1 ml of 70% ethanol, and protein contentwas quantitated by the Peterson assay (Peterson, 1979). The zeins werefound to comprise 45% of the sample dry weight, and so approximately 140mg of zein was obtained. Samples containing a range of 2.5 to 25 μg ofprotein were analyzed for purity and presence of the expected zeinprofile by SDS-PAGE and silver or Coomassie blue staining of the gels(Sambrook et al., 1989). The preparation displayed the expected proteinprofile, with the 27 kD, 19/22 kD, 16 kD, 15 kD, and 10 kD zeins allpresent in the expected proportions. This preparation was, therefore,used as the antigen in raising of polyclonal sera against total zeins.

The α-zeins (19/22 kD zeins) were extracted from the maize inbred lineA654 seed as follows: 6 grams of dry kernels were ground and processedas above for total zeins, from which approximately 500 mg of lyophilizedsample was obtained. After determining protein content, the zeins werefound to comprise 80% of the dry weight of the sample. To purify theα-zeins from the rest of the zeins, the sample was subjected topreparative SDS-PAGE: 10 mg of sample was weighed out, resuspended in500 μl of sample buffer/5% BME, then boiled for 10 minutes to eliminateaggregates before spinning for 30 seconds in the microfuge. Aliquots of55 μl/lane were run on a 3 mm thick gel with a 4% stacker and a 15%separation gel. Extra long plates (25 cm long by 14 cm wide) were usedto improve resolution. After running at 50 mA constant current for 3hours, the gel was run at 15 mA overnight. Proteins were visualized bystaining with cold 0.25 M TCA for approximately 10 minutes. Bands in the19/22 kD range were then excised and washed in SDS gel running bufferuntil the gel pieces appeared clear. This buffer was saved, gel pieceswere transferred to 2000 m.w. cutoff dialysis tubing. An additional 25mg of starting material was processed in this fashion also, and all gelslices were pooled before dialysis. The dialysis tubing was sealed withclips, and placed in a Biorad mini-sub gel apparatus with the clipsoriented perpendicularly with respect to the direction ofelectrophoresis. SDS running buffer was added to the level of thetubing, and elution was performed at 10 mA overnight. The electrodeswere reversed briefly, then the buffer inside the dialysis bag waspooled with the reserved buffer from the initial gel slice washes anddialyzed against 1 liter of deionized water, changing the water fivetimes over several hours. The dialysate was lyophilized, the protein wasquantitated, and then examined for purity by SDS-PAGE and silverstaining. No contaminating protein species were visible, and so thepurified antigen was used to inoculate rabbits for polyclonal antibodyproduction. The total amount of purified α-zein obtained from thisprocedure was 10.9 mg, resulting in a yield of 31% for the procedure.

B. Antigen Preparation and Injection

A total of six New Zealand white rabbits were used for antibodyproduction. Three were injected with purified α-zeins, and the remainingthree were injected with purified total zeins as described below. Two ofthe six rabbits were treated using the traditional Freund's complete andincomplete adjuvant, and the remaining four were treated with asynthetic adjuvant, as described below.

Both α- and total zeins were weighed, resuspended, and heated to 65° C.to completely solubilize the zeins; 0.5 mg of purified α-zein or 1.0 mgof total zein was resuspended in 60 μl of 70% ethanol for each rabbit tobe injected. Rabbits 1-3 received total zein as the antigen, and rabbits4-6 received purified α-zein antigen. For rabbits 1 and 4 (designated 1Fand 4F hereinafter), 440 μl of PBS/Tween (phosphate buffered saline/2%Tween 80, Sigma) was added to the zein solution, then 500 μl of Freund'scomplete adjuvant (Sigma) was added and the tubes were vortexedvigorously. The remaining four samples were made up as follows: to the60 μl of purified or total zein solution, 50 μl of AVRIDINE (a syntheticadjuvant from Kodak) made up in 100% ethanol to 140 mg/ml, 760 μl ofIntralipid 10% fat emulsion (Travenol), and 300 μl of PBS/Tween wereadded. After vortexing, the samples were sonicated in a cup sonicatorfor 2-30 second bursts (Ultrasonics, Inc.) to ensure complete emulsionbefore injection.

Samples were administered in 100 μl aliquots injected at multiple sitesacross the back of the animals. Boosts were administered every threeweeks, following the procedure above for formulating injection mixesexcept that Freund's incomplete adjuvant replaced the complete adjuvantfor rabbits 1F and 4F. A total of three boosts were administered, inaddition to the primary injection. Small volume (less than 5 ml) bleedswere performed to obtain sera for monitoring antibody titer andspecificity during the process. Specificity and titer of the antiserawere analyzed by running total zeins on SDS-PAGE/Western blots, asdescribed below. Once titers were found to be sufficient (reactive at a1:1000 sera dilution), several consecutive large (50 ml) bleeds wereperformed.

C. Analysis of Antisera

To determine antisera immunoreactivity and titer, total zein was assayedby SDS-PAGE/Western, with antisera dilutions from 1:50 to 1:1000 tested.The basic procedure was as follows: 500 ng of total zein/lane wasdissolved in 10 μl of sample buffer/2% BME, boiled 7.5 minutes, thenloaded on a 15% minigel (Mini Protean II, BioRad) with molecular weightmarkers (BRL) in alternate lanes, and run at 200 V for 45 minutes. Thestacking gel was removed, and the gel was equilibrated in transferbuffer (0.025 M Tris Cl, 0.194 M glycine, 20% methanol) for 10 minutesbefore being overlaid with a prepared membrane (Millipore Immobilon-P).Preparation of the membrane was performed by rinsing with methanol,according to the manufacturer's recommendations, before equilibrating intransfer buffer. Proteins were transferred at 27 V for 40 minutes n aGenie electroblotter (idea Scientific). After transfer, membranes wererinsed and blocked in 3% BSA/PBS for one hour at 37° C. on a shakerplatform. Membranes were divided into strips by cutting at lanescontaining molecular weight markers, and incubated with 10 ml of testantisera of the appropriate dilution overnight at 4° C., as well as withcontrol polyclonal antisera directed against total zein. After removalof the primary antisera, membrane strips were washed in 1×PBS, for 5×10minute washes, before incubation with the secondary antibody. Thesecondary antibody consisted of goat-anti-rabbit alkalinephosphatase-conjugated antibody (Kirkegaard-Perry Laboratories), diluted1:1000 in 3% BSA/PBS. After incubating for 1 hour at room temperaturewith shaking, strips were washed as above, and strips were incubated in4-chloro-napthol substrate solution (KPL) until color development wascomplete, approximately 2-5 minutes. Reactions were stopped by rinsingthe strips in deionized water.

The results showed that sera from all six rabbits displayed the expectedimmunoreactivity profiles. Specifically, sera from rabbits 1F, 2, and 3immunolabelled only the 19/22 kD zeins, and not the other zeins(indicating that the quality of the gel purified antigen was at least asgood as predicted by silver staining of SDS-PAGE, since antibodyproduction would actually be a more sensitive measurement ofcontamination with other protein species). In addition, the sera fromrabbits 4F, 5, and 6 exhibited reactivity with all of the zeins inapproximate proportion to the relative amounts of protein present in theprofile, showing slight to moderate labeling of the 27 kD zein, verystrong labeling of the abundant 19/22 kD zeins, moderate labeling of the16 and 14 kD zeins, and slight labeling of the less abundant 10 kD zein.

The titer of the antisera was also characterized by performinginmunolabelling of blots with dilutions ranging from 1:50 to 1:1000 (forlater bleeds). Although the lower dilutions of antisera immunolabelledthe same zeins as the corresponding sera at higher dilutions, backgroundstaining of the membrane increased at sera dilutions of less than 1:500.Since the expected immunoreactivity profiles (as discussed above) wereobtained at the 1:1000 dilution, this dilution was used for furtheranalyses. Testing of the sera at dilutions of 1:2000 and higher might beindicated if sera conservation is desired, since dilutions of more than1:1000 were not tested in these experiments.

The total amounts of sera obtained from the animals were as follows: 40ml each of sera from rabbits 1, 4, and 6, and 80 ml each of sera fromrabbits 2, 3, and 5. The latter rabbits were chosen for further bleedsbecause the immunoreactivity profiles appeared to be slightly morespecific to the α-zeins in the case of sera from rabbits 2 and 3 thanwas serum from rabbit 1F (which may have shown a very slight reactivitywith the 10 kD zein), and slightly more reactive with the 10 kD zein inthe case of sera from rabbits 5 and 6 than was sera from rabbit 4F.

EXAMPLE 5 Transformation of Maize with Z10 Promoter-Antisense Constructs

Embryogenic maize type II cultures were initiated from immature embryosisolated from developing seed derived from a cross of the genotypes B73and A188 as described in PCT publication WO 95/06128 and U.S.application Ser. No. 08/112,245. Type II cultures were microprojectilebombarded with a combination of plasmid vectors pDPG340 (Z10 promoter-Z4antisense DNA sequence, described above) or pDPG380 (Z10 promoter-A20antisense DNA sequence, described above) and pDPG363 comprising a plantexpression cassette containing the Cauliflower Mosaic Virus 35S promoteroperably linked in 5′ to 3′ order to intron 1 from the maize alcoholdehydrogenase I gene, the bar gene isolated from Streptomyceshygroscopicus, and the 3′ terminator and polyadenylation sequences fromthe nopaline synthase gene of Agrobacterium tumefaciens. Transformedcell lines were selected for resistance to the herbicide bialaphosconferred by expression of the bar gene as described in U.S. Pat. No.5,489,520, U.S. Pat. No. 5,550,318, and PCT publication WO 95/006128.Transformation of maize is further described in U.S. Pat. No. 5,538,877,U.S. Pat. No. 5,538,880, and PCT publication WO 95/06128, thedisclosures of which are incorporated by reference herein. Theidentification of transformed cell lines can be accomplished byemploying selectable or screenable markers, as described hereinabove.

The presence of the antisense DNA sequence in transformants was verifiedby polymerase chain reaction (PCR). The sequence of the 5′ PCR primerwas TCTAGGAAGCAAGGACACCACC (SEQ ID NO:5). The sequence of the 3′ PCRprimer was GCAAGACCGGCAACAGGATTCA (SEQ ID NO:6). The PCR reactionproduced a DNA fragment of size about 1.0 kilobases in transformantscontaining pDPG380 and a DNA fragment of about size 1.1 kilobases inpDPG340 transformants.

Transformed callus lines containing antisense DNA sequences operablylinked to a Z10 promoter were used to generate plants and seeds.Generally plants are regenerated as follows. Cells that survive theexposure to the selective agent, or cells that have been scored positivein a screening assay, were cultured in media that supports regenerationof plants. In an exemplary embodiment, the inventors modified MS and N6media (see Table 1 of U.S. application Ser. No. 08/594,861, thedisclosure of which is incorporated by reference herein) by includingfurther substances such as growth regulators. A preferred growthregulator for such purposes is dicamba or 2,4-D. However, other growthregulators may be employed, including NAA, NAA+2,4-C) or picloram. Mediaimprovement in these and like ways was found to facilitate the growth ofcells at specific developmental stages. Tissue was preferably maintainedon a basic media with growth regulators until sufficient tissue wasavailable to begin plant regeneration efforts, or following repeatedrounds of manual selection, until the morphology of the tissue issuitable for regeneration, at least two weeks, then transferred to mediaconducive to maturation of embryoids. Cultures were transferred everytwo weeks on this medium. Shoot development will signal the time totransfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, were then allowedto mature into plants. Developing plantlets were transferred to soillessplant growth mix, and hardened, e.g., in an environmentally controlledchamber at about 85% relative humidity, 600 ppm CO₂, and 25-250microeinsteins m⁻² s⁻¹ of light. Plants were preferably matured eitherin a growth chamber or greenhouse. Plants were regenerated from about 6weeks to 10 months after a transformant is identified, depending on theinitial tissue. During regeneration, cells were grown on solid media intissue culture vessels. Illustrative embodiments of such vessels werepetri dishes and Plant Con®s. Regenerating plants were preferably grownat about 19° to 28° C. After the regenerating plants reached the stageof shoot and root development, they were transferred to a greenhouse forfurther growth and testing.

By providing fertile, transgenic offspring, one can subsequently,through a series of breeding manipulations, move a selected gene fromone corn line into an entirely different corn line without the need forfurther recombinant manipulation. Movement of genes between corn linesis a basic tenet of the corn breeding industry, involving simply backcrossing the corn line having the desired gene (trait). Introducedtransgenes are valuable in that they behave genetically as any othercorn gene and can be manipulated by breeding techniques in a manneridentical to any other corn gene. Transformants containing Z10 promoterantisense constructs (pDPG340 and/or pDPG380) were crossed to variousmaize inbred lines, including elite inbred lines designated AW, CN, CV,and DD.

Zein proteins were extracted from mature kernels from a maize planttransformed with plasmids pDPG340 and pDPG380 and crossed to inbreds AWor CN, according to Tsai (1980), as follows. Fifty milligrams of groundkernel was suspended in 0.5 ml 70% ethanol, 1% β-mercaptoethanol andextracted at room temperature for 30 minutes to overnight. The samplewas vortexed, centrifuged at 12,000 rpm for 5 minutes. Fifty microlitersof the supernatant containing zein proteins was removed and dried. Zeinproteins were resuspended in 50 μl SDS polyacrylamide gel loading buffercontaining 1% β-mercaptoethanol. Protein was separated on SDSpolyacrylamide gels and stained with Coomassie blue. No qualitativedifferences were observed in the amounts of 19 kD and 22 kD α-zeinproteins (FIG. 5). Furthermore, overall protein expression in the kernelappears to be the similar in antisense transformants and untransformedmaize lines.

Analysis of the amino acid composition of Z10-antisense DNAtransformants was undertaken. Amino acids were extracted from maturekernels as described in Jarrett et al., 1986; Jones et al., 1983; AACC,1995). Results arc summarized in Table V. Data was analyzed by t-testsand differences noted between transformed and untransformed kernels thatwere significant at the p<0.05 level of significance. Transformed anduntransformed kernels are from the same ear. The level of leucine onlywas statistically significantly decreased in transformant DD021. Thelevel of lysine was statistically significantly increased and the levelof leucine statistically significantly decreased in transformants DD015and DD018. These results are expected if expression of α-zeins isdepressed in antisense transformants and expression of other proteins inthe endosperm are increased. α-Zein proteins are rich in leucineresidues and therefore one would expect that in the presence of reducedexpression of α-zein proteins, the level of leucine would decrease inthe kernel. Similarly, non-zein proteins contain more lysine than zeinproteins and therefore increased expression of non-zein proteins resultsin increased lysine levels in the kernel. Therefore, the amino acidcomposition data relating to Z10-antisense transformants is consistentwith a slight reduction in α-zein expression and increased expression ofnon-zein proteins, resulting in decreased levels of leucine andincreased levels of lysine in the seed. Similar increases in lysine anddecreases in leucine levels are observed in the maize opaque-2 mutantsin which zein synthesis is depressed and synthesis of non-zein proteinsis increased. Opaque-2 mutants, however, exhibit, other phenotypicdifferences from wild type maize (Di Fonzo et al., 1988; Bass et al.,1992).

TABLE V Trans- Lysine^(a,b) Leucine^(a,b) formant TransformedUntransformed Transformed Untransformed DD015 1.96* 1.75 11.68* 13.972.13 1.90 11.69* 14.50 DD021 2.40 2.09 15.90* 17.90 2.13 2.03 16.9717.75 DD038 1.96 2.00 12.66 13.89 1.82 1.96 15.87 15.73 DD018 2.74* 2.4317.57 19.13 2.30* 2.15 13.19* 15.52 ^(a)All amino acid concentrationsare expressed as milligrams amino acid per gram of seed. ^(b)Asteriskdenotes that amino acid concentration is statistically significantlydifferent from the amino acid concentration in an untransformed kernel.T-tests were performed to compare amino acid concentrations in isogenictransformed and untransformed kernels. Statistically significantdifferences are those for which p < 0.05.

EXAMPLE 6 Transformation of Maize with Z27 Promoter-Antisense ExpressionCassettes

Maize plants of the genotype A188×B73 were crossed to Hi-II maize plants(Armstrong et al., 1991). Immature embryos (1.2-2.0 mm in length) wereexcised from surface-sterilized, greenhouse-grown ears of Hi-II 11-12days post-pollination. The Hi-II genotype was developed from an A188×B73cross for high frequency development of type II callus from immatureembryos (Armstrong et al., 1991). Approximately 30 embryos per petridish were plated axis side down on a modified N6 medium containing 1mg/l 2,4-D, 100 mg/l casein hydrolysate, 6 mM L-proline, 0.5 g/l2-(N-morpholino)ethanesulfonic acid (MES), 0.75 g/l MgCl₂, and 2%sucrose solidified with 2 g/l Gelgro, pH 5.8 (#735 medium) Embryos werecultured in the dark for two to four days at 24° C.

Approximately four hours prior to bombardment, embryos were transferredto the above culture medium with the sucrose concentration increasedfrom 3% to 12%. When embryos were transferred to the high osmoticummedium they were arranged in concentric circles on the plate, starting 2cm from the center of the dish, positioned such that their coleorhizalend was orientated toward the center of the dish. Usually two concentriccircles were formed with 25-35 embryos per plate.

Gold particles were prepared containing 10 μg pDPG165 (described in U.S.Pat. No. 5,489,520), and 10 μg of pDPG530.

The plates containing embryos were placed on the third shelf from thebottom, 5 cm below the stopping screen in the bombardment chamber. The1100 psi rupture discs were used. Each plate of embryos was bombardedonce. Embryos were allowed to recover overnight on high osmotic strengthmedium prior to initiation of selection.

Embryos were allowed to recover on high osmoticum medium (735, 12%sucrose) overnight (16-24 hours) and were then transferred to selectionmedium containing 1 mg/l bialaphos (#739, 735 plus 1 mg/l bialaphos or#750, 735 plus 0.2M mannitol and 1 mg/l bialaphos). Embryos weremaintained in the dark at 24C. After three to four week on the initialselection plates about 90% of the embryos had formed Type II callus andwere transferred to selective medium containing 3 mg/l bialaphos (#758).Bialaphos resistant tissue was subcultured about every two weeks ontofresh selection medium (#758). Transformants were confirmed using PCRanalysis to detect presence of plasmid pDPG530. PCR primers used toconfirm presence of the Z27-antisense expression cassette in transformedtissue were as follows: 5′GCA CTT CTC CAT CAC CAC CAC 3′ (SEQ ID NO:6)and 5′TAT CCC CTT TCC AAC TTT CAG 3′ (SEQ ID NO:7). PCR amplification ofpDPG530 and pDPG531 transformants produced a DNA product of about 500base pairs.

Transformants were regenerated as generally described in PCT publicationWO 95/06128. Transformed embryogenic callus was transferred toregeneration culture medium (MS culture medium (Murashige and Skoog,1962), containing 0.91 mg/L L-asparagine, 1.4 g/L L-proline, 20 g/LD-sorbitol, 0.04 mg/L naphthalene acetic acid (NAA) and 3 mg/L6-benzylaminopurine). Cells were grown for about four weeks on thisculture medium with a transfer to fresh medium at about 2 weeks.Transformants were subsequently transferred to MS0 culture medium (MSmedium with no phytohormones added). Regenerated plants were transferredto soil as described previously in this application. Plants were crossedto maize inbred lines designated AW, CV, and DJ. Seed containing theZ27-antisense expression cassette were opaque in phenotype similar tokernels of opaque-2 mutant kernels. Furthermore, seed resulting fromcrosses of hemizygous Z-27-antisense transformants to untansformedinbreds resulted in seed segregating for the opaque phenotype incorrelation with the presence of the Z-27 antisense expression cassetteDNA sequence.

Zein proteins were extracted from mature kernels from maize plantstransformed with plasmids pDPG530 and crossed to inbreds AW or CV asfollows. Fifty milligrams of ground kernel was suspended in 0.5 ml 70%ethanol, 1% β-mercaptoethanol and extracted at room temperature for 30minutes to overnight. The sample was vortexed, centrifuged at 12,000 rpmfor 5 minutes. Fifty microliters of the supernatant containing zeinproteins was removed and dried. Zein proteins are resuspended in 50 μlSDS polyacrylamide gel loading buffer containing 1% β-mercaptoethanol.Protein was separated on SDS polyacrylamide gels and stained withCoomassie blue. Reduced amounts of 19 kD and 22 kD α-zeins were observedin five analyzed transformants. A Coomassie blue stained polyacrylamidegel of pDPG530 transformants and isogenic controls is shown in FIG. 6.In one transformant, designated KP014, expression of the 27 kD zeinprotein, a γ type zein protein was also depressed, suggesting thatexpression of an antisense DNA sequence in a maize may reduce expressionof a related family of genes, i.e., the α-zeins, but also a member of arelated family of proteins, i.e., 27 kD zein. A similar reduction in 27kD was also observed for sense DNA sequences (see FIG. 10). Isogeniccontrols were segregating kernels derived from plants lacking pDPG530DNA sequences, recovered from crosses of pDPG530 transformed plants tountransformed inbreds. Furthermore, overall protein expression in thekernel appears to be the greater in antisense transformants than inuntransformed maize lines as evidenced by overall protein staining byCoomassie blue on polyacrylamide gels (FIG. 7). Reduction of α-zeinsynthesis is observed in opaque-2 mutants, but the reduction is muchless than in Z4 antisense expressing maize transformants.

It is contemplated that antisense repression of zein protein synthesisin the seed is a result of reduction in the amount of zein RNA presentin the cell and consequently less synthesis of zein proteins. Northernblot analysis was completed to determine the level of steady state zeinRNA synthesis in pDPG580 transformants. Procedures for Northern blotanalysis are described in Sambrook et al. (1989). RNA isolated frommaize kernels 21 days after pollination was separated by agarose gelelectrophoresis and blotted to a Nitrobind membrane. The blot was probedwith the Z4 coding sequence. A Northern blot analysis of the KP015transformant is shown in FIG. 8. Darker signals on the autoradiograph,e.g., lanes 3, 9, and 14 (upper panel) and lanes 3, 5, 11, and 12 (lowerpanel), correspond to untransformed seeds which showed normal level ofzein synthesis. Other lanes (lighter signals) correspond to kernels thatshowed reduced levels of zein synthesis and the opaque phenotype inseeds containing the expression cassette.

Analysis of the amino acid composition of Z27-antisense DNAtransformants was undertaken. Amino acids were extracted from maturekernels derived from three independent transformed lines as follows.Fifty milligrams of ground corn meal was hydrolyzed in 1 ml 6N HCl underargon gas for 24 hours at 110° C. Samples were diluted to 50 ml andfiltered through a 0.45 micron filter. Norvaline as added to each sampleas an internal standard prior to HPLC analysis. Amino acids areseparated on a Supelcosil LC-8 HPLC column (Jarrett et al., 1986; Joneset al., 1983; AACC, 1995). Results from analysis of single kernels aresummarized in Table VI. Data was analyzed by t-tests and differencesnoted between transformed and untransformed kernels that weresignificant at the p<0.05 level of significance. Transformed anduntransformed kernels are isogenic segregants from a breedingpopulation. Lysine levels were statistically significantly increased inall kernels analyzed from the KP015 and KP016 transformants and lysinewas increased in four of six kernels analyzed from the KP014transformant. As expected leucine levels were decreased in mosttransformed kernels that were analyzed. These data demonstrate thatexpression of an antisense Z4 DNA sequence in transformed maize kernelscauses reduction in the quantities of α-zeins present in the kernel.Total protein in the antisense expressing kernel does not appear to bereduced. Furthermore, the observed decrease in α-zeins correlates withtransformed kernels with an opaque phenotype.

TABLE VI Trans- Lysine^(a,b) Leucine^(a,b) formant TransformedUntransformed Transformed Untransformed KP014 2.60* 2.08 13.94 16.852.85* 2.22 15.03* 17.08 3.09* 2.40 15.66 18.14 2.94* 2.45 15.27* 19.142.60 2.56 10.08 14.95 2.45* 2.08 9.21 10.58 KP015 1.90* 1.02 3.85* 7.801.92* 1.02 3.86* 7.98 1.48* 0.94 4.44* 5.87 1.43* 1.01 4.32* 6.26 KP0162.10* 1.52 8.58* 11.90 2.17* 1.54 8.95* 11.65 2.66* 2.03 14.16* 20.372.76* 1.81 14.68* 18.66 4.65* 2.14 11.01* 21.32 4.51* 2.31 11.26* 23.283.91* 2.22 12.96* 23.99 3.98* 2.36 13.29* 24.06 2.47* 1.76 9.60* 16.552.48* 1.70 9.70* 14.83 *Denotes differences from untransformed kernelsthat are statistically significant at the p < 0.05 level of confidence.

Endosperm cells in the maize kernel are comprised primarily of largestarch granules and protein sequestered in protein bodies (Lopes andLarkins, 1993). Zein proteins are essential for maintaining structure ofthe protein bodies (Lending and Larkins, 1989). A reduction in thenumber of protein bodies present in endosperm cells derived from a Z27promoter-antisense transformant was observed by light microscopy (FIG.9). This observation is further evidence that α-zein synthesis wasreduced in the Z27 promoter-antisense DNA transformants.

EXAMPLE 7 Transformation of Maize with Z27 Promoter-Sense ExpressionCassettes

In higher plants the phenomenon of co-suppression of gene expression hasbeen described (Napoli et al., 1990), Co-suppression refers to thesuppression of endogenous gene expression by expression of a transgenicsense DNA expression cassette. It was contemplated that a sense zeinexpression cassettes in maize may result in suppression of endogenouszein expression in a manner similar to that described in Example 6following expression of an antisense expression cassette.

Plasmid vector pDPG531 comprises a Z27 promoter-Z4 sense codingsequence-nopaline synthase 3′ region expression cassette. pDPG531differs from pDPG530 in that the Z4 coding sequence is operably linkedto the Z27 in the opposite orientation, i.e., pDPG531 is capable ofbeing transcribed and translated into the 22 kD zein protein. PlasmidpDPG531 and pDPG165 were introduced into maize cells as described inExample 6. Transformants were selected and regenerated as described inExample 6. Plants were regenerated from three Z27-Z4 sense expressioncassettes and crossed to inbreds designated AW, CV, and CN.

The amount of zein proteins present in untransformed and Z27-Z4 sensetransformants was compared on Coomassie blue stained polyacrylamide gelsas described previously in reference to analysis of antisensetransformants. Sample preparation and analysis were performed asdescribed in Example 6. FIG. 10 shows a Coomassie blue stainedpolyacrylamide gel. Each lane represents zein proteins extracted from asingle seed of a segregating population of untransformed and senseexpression cassette transformed seed. Lanes 1 through 8 represent seedderived from the transformant designated KQ012, and lanes 13 through 19represent seed derived from a second transformant designated KQ020.Lanes 9 through 12 represent untransformed maize seed. Lanes 3, 4, 7, 8,14, and 15 represent sense expression cassette transformed seed in whichthe α-zein levels are surprisingly greatly reduced in a mannercomparable to that observed in antisense transformants. In addition tothe unexpected reduction in zein protein concentration in sensetransformants, seed with reduced zein content also generally exhibit theopaque phenotype, and a reduction in Z27 zein levels.

In order to further determine whether the phenotype of Z27 promoter-Z4sense transformants was similar to antisense transformants, lysine andleucine concentrations were analyzed in seed derived from individualkernels. Amino acids were analyzed as described in Example 6. In onetransformant, designated KQ018, lysine and leucine levels werestatistically the same in isogenic transformed and untransformed seed.However, in a transformant designated KQ012, lysine levels werestatistically increased in the transformant and leucine levels werestatistically significantly decreased in the transformant. It istherefore apparent, that the Z27 promoter-Z4 sense transformants producea seed morphology, protein, and amino acid composition phenotype,similar to that observed in antisense transformants.

EXAMPLE 8 Method to Increase Content of Methionine in Plants

A method for increasing the methionine content of seeds involvescotransforming maize tissue culture with a zein sense or antisense DNAsequence (either A20 or Z4) and a DNA sequence containing a geneencoding a 10 kD zein protein. It is known that the 10 kD zein proteinsare rich in methionine. A decrease in expression of A20 and/or Z4 zeinproteins combined with an increase in expression in the 10 kD zeinproteins is likely to lead to about a 50% to 300% increase in totalweight percent of methionine in the seed.

Antisense or sense DNA sequences containing a DNA sequence complementaryor homologous to A20 and/or Z4 have been prepared as described inExamples 2 and 7. Conditions for successful transformation of maize celllines with the sense or antisense DNA sequence have been described inExamples 5 and 6.

A DNA sequence containing a gene encoding a 10 kD zein protein wasprepared as described in U.S. Pat. No. 5,508,468, which is herebyincorporated reference. Preferably, a Z10 DNA sequence contains a geneencoding a 10 kD zein protein including the 3′ noncoding sequencecombined with the promoter from a 27 kD zein protein. A plasmid withthis DNA sequence has been prepared and is designated pZ27Z10 and isdescribed in U.S. Pat. No. 5,508,468.

Transformed callus lines, plants, and seeds containing a DNA sequenceencoding a 10 kD zein protein were prepared as described in Examples 5and 6. Met1 seeds were generated as described in U.S. Pat. No.5,508,468.

The expression of the chimeric Z10 gene at the RNA level in Met1 seedswas demonstrated. Immature endosperms (21 DAP) were harvested from asegregating ear of the background Met1×A654 BC2. Both DNA and RNA wereprepared from individual endosperm samples. The DNA was analyzed by PCRfor the presence/absence of the Z27Z10 gene. The RNA samples wereanalyzed by Northern blot, probing with an oligonucleotide spanning thejunction between the Z27 promoter and the Z10 coding region. The resultsdemonstrate that the gene is expressed in endosperm tissue of PCR+seedsand not in that of PCR−seeds.

Seeds containing a DNA sequence containing the 10 kD zein proteincombined with the 27 kD promoter were field tested. A total of 130 earswere genotyped by PCR (using DNA from pooled leaf samples of germinatedseedlings) and analyzed for methionine content by amino acid analysis,and 10 kD zein levels by ELISA. There is a positive correlation between10 kD zein levels and methionine content in several maize backgroundstested. It is, therefore, contemplated that if α-zein synthesis isreduced by expression of sense or antisense zein constructs, expressionof a transgenic 10 kD zein will increase the methionine content of aseed. The results indicate that if it is possible to elevate theexpression of the 10 kD zein at least about 5-10 fold, methioninecontents in maize seed can be significantly raised (up to 2.5 to 3%).Additional transformants with the 10 kD zein functionally linked to the27 kD zein promoter and/or the Z422 kD zein promoter and/or the 10 kgzein promoter which show elevated levels of 10 kD zein and methionine intransformed seed have also been generated as described above and in U.S.Pat. No. 5,508,468.

Maize tissue cultures are cotransformed with a sense or antisense DNAsequence and a 10 kD zein DNA sequence and a selectable marker gene.Transformed cell lines containing both DNA sequences are identified byPCR analysis.

The transformed cell lines positive for PCR analysis for both anantisense and the 10 kD zein DNA sequences are used to regeneratetransformed plants and seeds, as described in Example 6. Seeds areanalyzed for expression of 10 kD zein and Z4 (22 kD) using Westernblots. Total methionine content of the seed is determined as describedin Examples 5 and 6.

An increase in the 10 kD zein expression combined with a decrease in theA20 and/or Z4 zein protein results in a significant increase (up toabout 50 to 300%) in the total methionine content of the seed.

EXAMPLE 9 Method to Increase Amino Acid Content of Particular AminoAcids in Seeds

The amino acid content of seeds is increased by expression of a geneencoding a synthetic polypeptide that comprises one or more amino acidsfor which altered levels are desired in the seed. Amino acid content isaltered by expression of a gene encoding a naturally occurring orsynthetic polypeptide comprising one or more desired amino acids, in aseed in which expression of endogenous seed storage proteins have beenrepressed by expression of a sense or antisense seed storage protein DNAsequence.

For example, a gene encoding the synthetic protein MB1 is introducedinto a plant in which storage protein synthesis is repressed byexpression of a sense or antisense DNA sequence. The MB1 coding sequenceis introduced into a transgenic plant with reduced expression of storageprotein, wherein said plant was previously transformed with a storageprotein sense or antisense DNA sequence. Alternatively, the MB1 sequenceis transformed into a plant simultaneously with a storage protein senseor antisense DNA sequence. In a preferred embodiment of the presentinvention, a storage protein antisense or sense expression cassette andan MB1 expression cassette are transformed into maize simultaneously orsequentially as described in Examples 5, 7, and 8.

A plasmid vector, designated pDPG780, containing an MB1 plant expressioncassette was constructed. The MB1 protein coding sequence was obtainedfrom Mary A. Hefford (Center for Food and Animal Research, Agricultureand Agri-Food Canada, Ottawa, ON, K1A 0C6, Canada) and the DNA sequenceis disclosed in Beauregard et al., 1995. MB1 is a synthetic proteinenriched in methionine, threonine, lysine and leucine and exhibitsα-helical structure similar to a zein protein. Plasmid vector pDPG780was constructed by operably linking an endoplasmic reticulum signalsequence (Pedersen et al., 1986) from the 15 kD zein protein encodinggene 5′ to the MB1 coding sequence. The 15 kD zein-MB1 sequence wasinserted in plasmid vector pZ27-nos between the Z27 promoter element andthe nopaline synthase 3′ region (nos). The expression cassette comprisesin 5′ to 3′ orientation, the Z27 promoter, Z15 signal sequence, MB1coding sequence, and nos 3′ region. One of skill in the art couldconstruct addition plasmid vectors containing a seed specific promoteroperably linked to an endoplasmic reticulum signal sequence, proteinencoding sequence, and 3′ region, wherein said protein encoding sequencecomprises a DNA sequence encoding a protein of desired amino acidcomposition.

The plasmid vector pDPG780 is introduced into maize in conjunction witha vector comprising a selectable marker gene, e.g., pDPG165 comprisingthe bar gene. The MB1 expression cassette is transformed into maizeplants containing a sense or antisense zein transgene in which synthesisof α-zein proteins is repressed. Alternatively, the sense or antisensezein construct is transformed into maize simultaneously with the MB1expression cassette.

Plants are regenerated as described in Examples 5, 6 and 7. Proteincomposition of seed is analyzed by polyacrylamide gel electrophoresis asdescribed in Examples 5 and 6. Reduction in zein proteins is observedand expression of a protein of desired amino acid composition isobserved. Amino acid composition of seed is determined as described inExamples 5, 6, and 7. Levels of desired amino acids are altered inaccordance with the amino acid composition of the protein encoded by thetransgene.

While the present invention has been described in connection with thepreferred embodiment thereof, it will be understood many modificationswill be readily apparent to those skilled in the art, and thisapplication is intended to cover any adaptations or variations thereof.All patents, patent documents and publications described herein arehereby incorporated by reference.

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1. A fertile transgenic Zea mays plant comprising seeds having anincreased starch extractability comprising a nucleic acid moleculeexhibiting at least 70% sequence identity to SEQ ID NO: 1 or 90%sequence identity to SEQ ID NO:2, wherein SEQ ID NO: 1 encodes a 19 kDα-zein plant seed storage protein and SEQ ID NO:2 encodes a 22 kD α-zeinplant seed storage protein and wherein said nucleic acid molecule isoperably linked to a promoter in sense or antisense orientation, whereintranscription of said nucleic acid molecule inhibits expression of saidseed storage protein and increases the starch extractability of the seedrelative to the amount of said seed storage protein and starchextractability of seeds not comprising said nucleic acid molecule.
 2. Aseed from the plant of claim 1, wherein the seed comprises said nucleicacid molecule.
 3. A progeny plant produced from the seed of claim 2,wherein the plant comprises said nucleic acid molecule and whereintranscription of said nucleic acid molecule inhibits expression of saidseed storage protein and increases the starch extractability of theprogeny plant's seed relative to the amount of said seed storage proteinand starch extractability of seeds not comprising said nucleic acidmolecule.
 4. The transgenic plant of claim 1, wherein the promotercomprises the 10 kD zein promoter.
 5. The transgenic plant of claim 1,wherein the promoter comprises the 27 kD zein promoter.
 6. Thetransgenic plant of claim 1, wherein the nucleic acid molecule is inantisense orientation and when transcribed produces a mRNA moleculecomplementary to a mRNA encoding 19 kD α-zein protein.
 7. Thc transgenicplant of claim 1, wherein the nucleic acid molecule is in antisenseorientation and when transcribed produces a mRNA molecule complementaryto a mRNA encoding 22 kD α-zein protein.
 8. The transgenic plant ofclaim 2, or 3, further comprising at least one selectable marker gene.9. A method of obtaining starch from a Zea mays seed, comprising: (a)growing a transgenic Zea mays plant, comprising a nucleic acid moleculeexhibiting at least 70% sequence identity to SEQ ID NO: 1 or 90%sequence identity to SEQ ID NO:2, wherein SEQ ID NO: 1 encodes a 19 kDα-zein plant seed storage protein and SEQ ID NO:2 encodes a 22 kD α-zeinplant seed storage protein and wherein said nucleic acid molecule isoperably linked to a promoter in sense or antisense orientation whereintranscription of said nucleic acid molecule inhibits expression of saidseed storage protein and increases the starch extractability of the seedrelative to the amount of said seed storage protein and starchextractability of seeds not comprising said nucleic acid molecule; (b)obtaining seed from said plant; and (c) extracting starch from the seed.10. The method of claim 9 wherein the nucleic acid molecule is operablylinked to a promoter functional in plant cells.
 11. The method of claim10 wherein the promoter comprises the 10 kD zein promoter.
 12. Themethod of claim 10 wherein the promoter comprises the 27 kD zeinpromoter.
 13. The method of claim 9 wherein the nucleic acid molecule isin sense orientation.
 14. The method of claim 9 wherein the nucleic acidmolecule is in antisense orientation.
 15. The method of claim 13 whereinthe nucleic acid molecule exhibiting at least 70% sequence identity withSEQ ID NO: 1 is operably linked to the promoter in sense orientation.16. The method of claim 13 wherein the nucleic acid molecule exhibitingat least 90% sequence identity with SEQ ID NO:2 is operably linked tothe promoter in sense orientation.
 17. The method of claim 14 whereinthe nucleic acid molecule exhibiting at least 70% sequence identity withSEQ ID NO: 1 is operably linked to the promoter in antisenseorientation.
 18. The method of claim 14 wherein the nucleic acidmolecule exhibiting at least 90% sequence identity with SEQ ID NO:2 isoperably linked to the promoter in antisense orientation.
 19. The methodof claim 9 wherein the genome of the transgenic Zea mays plant furthercomprises at least one selectable marker gene.